Phototherapeutic light for treatment of pathogens

ABSTRACT

Methods and related devices for impinging light on tissue, for example within a body of a patient, to induce various biological effects are disclosed. Biological effects may include at least one of inactivating and/or inhibiting growth of one or more pathogens, upregulating a local immune response, stimulating enzymatic generation of nitric oxide to increase endogenous stores of nitric oxide, releasing nitric oxide from endogenous stores of nitric oxide, and inducing an anti-inflammatory effect. Wavelengths of light are selected based on intended biological effects for one or more of targeted tissue types and targeted pathogens. Light treatments may provide multiple pathogenic biological effects, either with light of a single wavelength or with light having multiple wavelengths. Devices and methods for light treatments are disclosed that provide light doses for inducing biological effects on various targeted pathogens and targeted tissues with increased efficacy and reduced cytotoxicity.

STATEMENT OF RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.17/117,858, filed Dec. 10, 2020, which is a continuation-in-part of U.S.patent application Ser. No. 16/898,385, filed Jun. 10, 2020; which is acontinuation of U.S. patent application Ser. No. 16/709,550, filed onDec. 10, 2019; which is a continuation of U.S. patent application Ser.No. 15/222,199, filed on Jul. 28, 2016, now U.S. Pat. No. 10,525,275;which claims the benefit of provisional patent application Ser. No.62/197,746, filed Jul. 28, 2015, the disclosures of which are herebyincorporated herein by reference in their entireties.

U.S. patent application Ser. No. 17/117,858 claims the benefit ofprovisional patent application Ser. No. 63/123,631, filed Dec. 10, 2020;provisional patent application Ser. No. 63/084,802, filed Sep. 29, 2020;provisional patent application Ser. No. 63/074,800, filed Sep. 4, 2020;and provisional patent application Ser. No. 62/987,318, filed Mar. 9,2020, the disclosures of which are hereby incorporated herein byreference in their entireties.

TECHNICAL FIELD

The present disclosed subject matter relates generally to devices andmethods for impinging light on tissue (e.g., phototherapy or lighttherapy) to induce one or more biological effects. Additionally,disclosed are methods and devices for delivering light as a therapeutictreatment for tissue that comes into contact with or is infected bypathogens. This disclosure additionally relates to systems and methodsfor phototherapeutic stimulation of nitric oxide production and/orrelease in tissues of mammalian subjects.

BACKGROUND

The term “phototherapy” relates to the therapeutic use of light. Variouslight therapies (e.g., including low level light therapy (LLLT) andphotodynamic therapy (PDT)) have been publicly reported or claimed toprovide various health related medical benefits—including, but notlimited to: promoting hair growth; treatment of skin or tissueinflammation; promoting tissue or skin healing or rejuvenation;enhancing wound healing; pain management; reduction of wrinkles, scars,stretch marks, varicose veins, and spider veins; treating cardiovasculardisease; treating erectile dysfunction; treating microbial infections;treating hyperbilirubinemia; and treating various oncological andnon-oncological diseases or disorders.

Various mechanisms by which phototherapy has been suggested to providetherapeutic benefits include: increasing circulation (e.g., byincreasing formation of new capillaries); stimulating the production ofcollagen; stimulating the release of adenosine triphosphate (ATP);enhancing porphyrin production; reducing excitability of nervous systemtissues; modulating fibroblast activity; increasing phagocytosis;inducing thermal effects; stimulating tissue granulation and connectivetissue projections; reducing inflammation; and stimulating acetylcholinerelease.

Phototherapy has also been suggested to stimulate cells to generatenitric oxide. Various biological functions attributed to nitric oxideinclude roles as signaling messenger, cytotoxin, antiapoptotic agent,antioxidant, and regulator of microcirculation. Nitric oxide isrecognized to relax vascular smooth muscles, dilate blood vessels,inhibit aggregation of platelets, and modulate T cell-mediate immuneresponse.

Nitric oxide is produced by multiple cell types in tissue, and is formedby the conversion of the amino acid L-arginine to L-citrulline andnitric oxide, mediated by the enzymatic action of nitric oxide synthases(NOSs). NOS is a NADPH-dependent enzyme that catalyzes the followingreaction:

L-arginine+3/2 NADPH+H⁺+2 O₂

citrulline+nitric oxide+3/2 NADP⁺

In mammals, three distinct genes encode NOS isozymes: neuronal (nNOS orNOS-I), cytokine-inducible (iNOS or NOS-II), and endothelial (eNOS orNOS-III). iNOS and nNOS are soluble and found predominantly in thecytosol, while eNOS is membrane associated. Many cells in mammalssynthesize iNOS in response to inflammatory conditions.

Skin has been documented to upregulate inducible nitric oxide synthaseexpression and subsequent production of nitric oxide in response toirradiation stress. Nitric oxide serves a predominantly anti-oxidantrole in the high levels generated in response to radiation.

Nitric oxide is a free radical capable of diffusing across membranes andinto various tissues; however, it is very reactive, with a half-life ofonly a few seconds. Due to its unstable nature, nitric oxide rapidlyreacts with other molecules to form more stable products. For example,in the blood, nitric oxide rapidly oxidizes to nitrite, and is thenfurther oxidized with oxyhaemoglobin to produce nitrate. Nitric oxidealso reacts directly with oxyhaemoglobin to produce methaemoglobin andnitrate. Nitric oxide is also endogenously stored on a variety ofnitrosated biochemical structures including nitrosoglutathione (GSNO),nitrosoalbumin, nitrosohemoglobin, and a large number of nitrosocysteineresidues on other critical blood/tissue proteins. The term “nitroso” isdefined as a nitrosated compound (nitrosothiols (RSNO) or nitrosamines(RNNO)), via either S- or N-nitrosation. Examples of nitrosatedcompounds include GSNO, nitrosoalbumin, nitrosohemoglobin, and proteinswith nitrosated cysteine residue. Metal nitrosyl (M-NO) complexes areanother endogenous store of circulating nitric oxide, most commonlyfound as ferrous nitrosyl complexes in the body; however, metal nitrosylcomplexes are not restricted to complexes with iron-containing metalcenters, since nitrosation also occurs at heme groups and coppercenters. Examples of metal nitrosyl complexes include cytochrome coxidase (CCO-NO) (exhibiting 2 heme and 2 copper binding sites),cytochrome c (exhibiting heme center binding), and nitrosylhemoglobin(exhibiting heme center binding for hemoglobin and methemoglobin),embodying endogenous stores of nitric oxide.

FIG. 1 is a reaction sequence showing photoactivated production ofnitric oxide catalyzed by iNOS, followed by binding of nitric oxide toCCO.

When nitric oxide is auto-oxidized into nitrosative intermediates, thenitric oxide is bound covalently in the body (in a “bound” state). Thus,conventional efforts to produce nitric oxide in tissue may have alimited therapeutic effect, since nitric oxide in its “gaseous” state isshort-lived, and cells being stimulated to produce nitric oxide maybecome depleted of NADPH or L-Arginine to sustain nitric oxideproduction.

Viral infections pose a great challenge to human health, particularlyrespiratory tract infections of the Orthomyxoviridae (e.g. influenza)and Coronaviridae (e.g. SARS-CoV-2) families. Additionally, DNA virusesincluding the Papovaviridae family (e.g. human papillomavirus (HPV))have extremely wide prevalence that result in low risk papillomas of theskin and high risk papillomas of mucosal epithelial tissue. Infection bythe human papillomavirus (HPV) is currently the most common sexuallytransmitted disease (STD). While most HPV infections are asymptomaticand resolve without treatment, some infections result in warts orprecancerous lesions. The presence and persistence of precancerouslesions increase the risk for a cancer developing, particularly in thecervix, vulva, vagina, penis, anus, mouth, or throat. The genotype ofthe HPV is significant as HPV type 16 and HPV type 18 appear to causeabout 70% of cervical cancer cases. Furthermore, up to 90% of the othercancers are also linked to HPV. Fifteen HPV types are currently believedto be responsible for all cervical cancers. While HPV type 16 is mostcommonly associated with cervical precancerous lesions and cancerouslesions, HPV types 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68,73, and 82 are also implicated in cervical cancer. When affecting thecervix, the precancerous lesions are referred to as cervical dysplasia.It is estimated that there are around 500,000 patients per year in theUnited States surgically treated for cervical dysplasia. Conventionalmanagement of cervical dysplasia calls for colposcopy with endocervicalsampling which allows the dysplasia to be rated as cervicalintraepithelial neoplasia I, II or III (CIN I, II, or III). With CIN Iand a satisfactory colposcopy, one approach is to “watch and wait” todetermine whether the condition worsens over 6 months to a year asdetermined by colposcopy. Another approach is to perform an invasive(e.g. surgical) treatment involving the cervix. Commonly used treatmentmethods include medication, electro-cauterization, cryosurgery, laservaporization, and surgery. Cryotherapy involves cooling cervical tissueto sub-zero temperature which results in freezing. While simple andrelatively inexpensive, abnormal cells below the surface are untreatedmaking the approach unsuitable for large or severe dysplasia. Loopexcision, LEEP (loop electrosurgical excision procedure), is a treatmentthat uses a loop of wire to remove infected tissue. The wire loop iselectrically energized to facilitate removal of abnormal portions of thecervix. Cramping and bleeding are common side-effects. A cone biopsyinvolves removal of tissue from the cervix and the endocervical canal,performed conventionally or using a laser. Bleeding and pain are commonafter the procedure, which is typically done under anesthesia. Ahysterectomy may also be done to resolve the infection, but it is amajor surgical procedure unsuitable for women who wish to becomepregnant in the future. The goal of these procedures is to remove thoseabnormal cells from the cervix. Cervical cancer has been reported tohave a global survival rate of about 52%. A non-surgical treatment thatreduced or eradicated HPV viral infections could have a significantimpact on women's health.

SUMMARY

Aspects of the present disclosure relate to devices and methods forimpinging light on a tissue, for example within a body of a patient,where the light may include at least one characteristic that exerts orinduces at least one biological effect within or on the tissue.Biological effects may include at least one of inactivating andinhibiting growth of one or more combinations of microorganisms andpathogens, including but not limited to viruses, bacteria, fungi, andother microbes, among others. Biological effects may also include one ormore of upregulating a local immune response, stimulating enzymaticgeneration of nitric oxide to increase endogenous stores of nitricoxide, releasing nitric oxide from endogenous stores of nitric oxide,and inducing an anti-inflammatory effect. Wavelengths of light may beselected based on at least one intended biological effect for one ormore of the targeted tissue and the targeted microorganisms orpathogens. In certain aspects, wavelengths of light may include visiblelight in any number of wavelength ranges based on the intendedbiological effect. Further aspects involve light impingement on tissuefor multiple microorganisms and/or multiple pathogenic biologicaleffects, either with light of a single peak wavelength or a combinationof light with more than one peak wavelength. Devices and methods forlight treatments are disclosed that provide light doses for inducingbiological effects on various targeted pathogens and targeted tissueswith increased efficacy and reduced cytotoxicity. Light doses mayinclude various combinations of irradiances, wavelengths, and exposuretimes, and such light doses may be administered continuously ordiscontinuously with a number of pulsed exposures.

Because of the relative costs, both economically and on the health andwell-being of patients, new treatments to inhibit or eradicate viralinfections in tissues, particularly the mucosal epithelial surfaces likethe cervix, mouth, nose, throat and anus, are greatly needed. Suchtreatments and devices therefore are provided for herein.

Phototherapy has attracted significant attention as a therapeutictreatment for various maladies and conditions. Devices for deliveringphototherapy to inhibit or eradicate viral infections and methods ofusing the same are disclosed herein. Irradiances of light represented inmilliwatts per square centimeter (mW/cm²) have been proposed at aspecific wavelength for a threshold time over a given duration to yieldtherapeutic dosages represented in joules per square centimeter (J/cm²)which are effective for inactivating viruses or treating viralinfections while maintaining the viability of epithelial tissues. Thesetreatments can be tailored to the particular tissue being treated, aswell as to the various fluids in the media, such as blood, sputum,saliva, cervical fluid, and mucous. The total dosage (J/cm²) to treat aninfection can be spread out over multiple administrations, with eachdose applied over seconds or minutes, and with multiple doses over daysor weeks, at individual doses that treat the infection while minimizingdamage to the particular tissue.

Certain aspects of the disclosure relate to phototherapeutic modulationof nitric oxide in living mammalian tissue, including use of lighthaving a first peak wavelength and a first radiant flux to releasenitric oxide from endogenous stores of nitric oxide, and use of lighthaving a second peak wavelength and a second radiant flux to stimulateenzymatic generation of nitric oxide to increase endogenous stores ofnitric oxide, wherein the second peak wavelength differs from the firstpeak wavelength.

In a first aspect, the disclosure relates to a method of modulatingnitric oxide in living mammalian tissue. The method includes impinginglight having a first peak wavelength on the tissue at a first radiantflux, wherein the first peak wavelength and the first radiant flux areselected to stimulate enzymatic generation of nitric oxide to increaseendogenous stores of nitric oxide. The method further includes impinginglight having a second peak wavelength on the tissue at a second radiantflux, wherein the second peak wavelength and the second radiant flux areselected to release nitric oxide from the endogenous stores, wherein thesecond peak wavelength is greater than the first peak wavelength by atleast 25 nm, by at least 50 nm, or another threshold specified herein.In certain embodiments, each of the first radiant flux and the secondradiant flux is in a range of from 5 mW to 60 mW.

In certain embodiments, the enzymatic generation of nitric oxide ismediated by iNOS, nNOS, and/or eNOS in or proximate to the tissue. Incertain embodiments, the endogenous stores of nitric oxide comprisenitrosoglutathione, nitrosoalbumin, nitrosohemoglobin, nitrosothiols,nitrosamines, and/or metal nitrosyl complexes in or proximate to thetissue.

In certain embodiments, the method further includes sensing atemperature condition on or proximate to (a) a therapeutic devicearranged to emit at least one of the light having the first peakwavelength or the light having the second peak wavelength, or (b) thetissue; generating at least one signal indicative of the temperaturecondition; and controlling at least one of the following items (i) or(ii) responsive to the at least one signal: (i) impingement of lighthaving the first peak wavelength on the tissue, or (ii) impingement oflight having the second peak wavelength on the tissue.

In another aspect, the disclosure relates to a device for modulatingnitric oxide in living mammalian tissue. The device includes means forimpinging light having a first peak wavelength on the tissue at a firstradiant flux, wherein the first peak wavelength and the first radiantflux are selected to stimulate enzymatic generation of nitric oxide toincrease endogenous stores of nitric oxide. The device further includesmeans for impinging light having a second peak wavelength on the tissueat a second radiant flux, wherein the second peak wavelength and thesecond radiant flux are selected to release nitric oxide from theendogenous stores, wherein the second peak wavelength is greater thanthe first peak wavelength by at least 25 nm.

In certain embodiments, the device further includes means for sensing atemperature condition on or proximate to (a) the device or (b) thetissue; means for generating at least one signal indicative of thetemperature condition; and means for controlling at least one of thefollowing items (i) or (ii) responsive to the at least one signal: (i)impingement of light having the first peak wavelength on the tissue, or(ii) impingement of light having the second peak wavelength on thetissue.

In another aspect, the disclosure relates to another device formodulating nitric oxide in living mammalian tissue. The device includesat least one first light emitting device configured to impinge lighthaving a first peak wavelength on the tissue at a first radiant flux,wherein the first peak wavelength and the first radiant flux areselected to release nitric oxide from endogenous stores of nitric oxide.The device further includes at least one second light emitting deviceconfigured to impinge light having a second peak wavelength on thetissue at a second radiant flux, wherein the second peak wavelength andthe second radiant flux are selected to stimulate enzymatic generationof nitric oxide to increase endogenous stores of nitric oxide, whereinthe second peak wavelength exceeds the first peak wavelength by at least25 nm, at least 50 nm, or another threshold specified herein. In certainembodiments, the device further includes driver circuitry configured todrive the at least one first light emitting device and the at least onesecond light emitting device. In certain embodiments, each of the firstradiant flux and the second radiant flux is in a range of from 5 mW to60 mW.

In certain embodiments, the device further includes at least one thirdlight emitting device configured to impinge light having a third peakwavelength on the tissue, wherein the third peak wavelength differs fromeach of the first peak wavelength and the second peak wavelength by atleast 10 nm.

In certain embodiments, the device further includes a temperature sensorarranged to sense a temperature condition on or proximate to at leastone of (a) a portion of the device or (b) the tissue, wherein at leastone of initiation of operation, deviation of operation, or terminationof operation of any of (i) the at least one first light emitting deviceor (ii) the at least one second light emitting device is responsive toan output signal of the temperature sensor.

In certain embodiments, the device further includes a flexible substratesupporting the at least one first light emitting device and the at leastone second light emitting device.

In certain embodiments, the device further includes a light-transmissive(e.g., encapsulant) material layer covering the at least one first lightemitting device, the at least one second light emitting device, and atleast a portion of the flexible substrate.

In certain embodiments, the device further includes a plurality of holesdefined in the flexible substrate and the light-transmissive materiallayer, wherein the plurality of holes are arranged to permit transittherethrough of at least one of air, vapor, or exudate.

In certain embodiments, the device is configured to contact, beconnected to, or conform to a skin or other tissue of a patient with atleast a portion of the light-transmissive material layer arranged incontact with the skin or other tissue of the patient. In otherembodiments, the device is configured to be spatially separated from atargeted irradiation area, such as being arranged not to contact tissueof the patient.

In certain embodiments, the device further includes a substantiallyrigid substrate supporting the at least one first light emitting deviceand the at least one second light emitting device, wherein at least aportion of the device is configured for insertion into a body cavity ofa patient.

In certain embodiments, the device further includes at least onewaveguide arranged between (i) the tissue and (ii) at least one of theat least one first light emitting device or the at least one secondlight emitting device.

In certain embodiments, the device further includes a light scatteringmaterial, a textured light scattering surface, or a patterned lightscattering surface arranged between (i) the tissue and (ii) at least oneof the at least one first light emitting device or the at least onesecond light emitting device.

In certain embodiments, the device further includes an energy storageelement arranged to supply power to the driver circuitry.

In another aspect, the disclosure relates to a device for deliveringlight energy to tissue of a patient. The device includes at least onefirst solid state light emitting device configured to impinge lighthaving a first peak wavelength on the tissue. The device furtherincludes at least one second solid state light emitting deviceconfigured to impinge light having a second peak wavelength on thetissue. The device additionally includes driver circuitry configured todrive the at least one first solid state light emitting device and theat least one second solid state light emitting device. The first peakwavelength and the second peak wavelength are selected from one of thefollowing combinations (a) to (g): (a) the first peak wavelength is in arange of from 410 nm to 490 nm and the second peak wavelength is in arange of from 500 nm to 600 nm; (b) the first peak wavelength is in arange of from 620 nm to 640 nm and the second peak wavelength is in arange of from 650 nm to 670 nm; (c) the first peak wavelength is in arange of from 520 nm to 540 nm and the second peak wavelength is in arange of from 650 nm to 670 nm; (d) the first peak wavelength is in arange of from 400 nm to 420 nm and the second peak wavelength is in arange of from 620 nm to 640 nm; (e) the first peak wavelength is in arange of from 400 nm to 420 nm and the second peak wavelength is in arange of from 650 nm to 670 nm; (f) the first peak wavelength is in arange of from 400 nm to 420 nm and the second peak wavelength is in arange of from 495 nm to 515 nm; or (g) the first peak wavelength is in arange of from 400 nm to 420 nm and the second peak wavelength is in arange of from 516 nm to 545 nm. In certain embodiments, the first peakwavelength is in a range of from 400 nm to 420 nm and the second peakwavelength is in a range of from 525 nm to 535 nm.

In certain embodiments, the device further includes a temperature sensorarranged to sense a temperature condition on or proximate to at leastone of (a) a portion of the device or (b) the tissue, wherein at leastone of initiation of operation, deviation of operation, or terminationof operation of at least one of (i) the at least one first solid statelight emitting device or (ii) the at least one second solid state lightemitting device is responsive to an output signal of the temperaturesensor.

In another aspect, the disclosure relates to a method of modulatingnitric oxide in living mammalian tissue, the method comprising:impinging light on the tissue, wherein the light impinged on the tissuecomprises incoherent light emissions including a first peak wavelengthin a range of from 410 nm to 440 nm and a first radiant flux, andwherein the first peak wavelength and the first radiant flux areselected to stimulate at least one of (i) enzymatic generation of nitricoxide to increase endogenous stores of nitric oxide or (ii) release ofnitric oxide from endogenous stores of nitric oxide; wherein the lightimpinged on the tissue is substantially devoid of light emissions havinga peak wavelength in a range of from 600 nm to 900 nm.

In certain embodiments, the light impinged on the tissue is devoid ofemissions of any wavelength conversion material stimulated by incoherentlight emissions including a first peak wavelength in a range of from 410nm to 440 nm. In certain embodiments, the tissue is devoid of an appliedor received photosensitive therapeutic compound or agent. In certainembodiments, at least 65% (or at least 80%, or at least 90%) of afluence of light impinged on the tissue consists of the incoherent lightemissions including a first peak wavelength in a range of from 410 to440 nm. In certain embodiments, the light impinged on the tissue issubstantially devoid of light emissions having a peak wavelength in arange of from 441 nm to 490 nm. In certain embodiments, the incoherentlight emissions including a first peak wavelength in a range of from 410nm to 440 nm are provided as a plurality of discrete pulses. In certainembodiments, the light impinged on the tissue further comprisesincoherent light emissions including a second peak wavelength in a rangeof from 500 nm to 540 nm. In certain embodiments, the incoherent lightemissions including a first peak wavelength in a range of from 410 nm to440 nm are impinged on the tissue during a first time window, theincoherent light emissions including a second peak wavelength in a rangeof from 500 nm to 540 nm are impinged on the tissue during a second timewindow, and at least a portion of the second time window isnon-overlapping with the first time window. In certain embodiments, thefirst peak wavelength and the first radiant flux are selected to releaseendogenous stores of nitric oxide. In certain embodiments, the secondpeak wavelength and the second radiant flux are selected to stimulateenzymatic generation of nitric oxide to increase endogenous stores ofnitric oxide. In certain embodiments, the tissue comprises at least oneof epithelial tissue, mucosal tissue, bone, connective tissue, muscletissue, or cervical tissue. In certain embodiments, the tissue comprisesdermal tissue. In certain embodiments, a method further comprisessensing a temperature condition on or proximate to (a) a therapeuticdevice arranged to impinge light on the tissue, or (b) the tissue;generating at least one signal indicative of the temperature condition;and controlling impingement of light on the tissue responsive to the atleast one signal. In certain embodiments, the light impinged on thetissue comprises a fluence of from about 0.5 J/cm² to about 100 J/cm²,or from about 5 J/cm² to about 50 J/cm².

In another aspect, the disclosure relates to a device for modulatingnitric oxide in living mammalian tissue, the device comprising: anambient light blocking element; and at least one first light emittingelement positioned between the ambient light blocking element and thetissue, wherein the at least one first light emitting element isconfigured to impinge incoherent light on the tissue, said incoherentlight having a first peak wavelength and a first radiant flux, whereinthe first peak wavelength and the first radiant flux are selected tostimulate at least one of (i) enzymatic generation of nitric oxide toincrease endogenous stores of nitric oxide or (ii) release of nitricoxide from endogenous stores of nitric oxide; wherein the device issubstantially devoid of any light emitting element configured to impingelight on the tissue, said light having a peak wavelength in a range offrom 600 nm to 900 nm.

In certain embodiments, the device is substantially devoid of any lightemitting element configured to impinge light having a peak wavelength ina range of from 441 nm to 490 nm on the tissue. In certain embodiments,the device is devoid of any wavelength conversion material configured tobe stimulated by the at least one first light emitting element. Incertain embodiments, the device further comprises a flexible substratesupporting the at least one first light emitting element. In certainembodiments, the device is configured to contact, be connected to, orconform to the tissue with a light-transmissive material. In certainembodiments, light impinged on the tissue is substantially devoid oflight emissions having a peak wavelength in a range of from 441 nm to490 nm. In certain embodiments, the device further comprises drivercircuitry configured to generate incoherent light emissions includingthe first peak wavelength, wherein the first peak wavelength is in arange of from 410 nm to 440 nm, and said incoherent light emissionscomprise a plurality of discrete pulses.

In certain embodiments, the device further comprises at least one secondlight emitting element configured to impinge incoherent light on thetissue, said incoherent light having a second peak wavelength and asecond radiant flux, wherein the second peak wavelength is in a range offrom 500 nm to 540 nm. In certain embodiments, the device is configuredto impinge incoherent light emissions including the first peakwavelength during a first time window, wherein the first peak wavelengthis in a range of from 410 nm to 440 nm, and being configured to impingeincoherent light emissions including the second peak wavelength in arange of from 500 nm to 540 nm during a second time window, wherein atleast a portion of the second time window is non-overlapping with thefirst time window. In certain embodiments, the device further comprisesa probe configured for insertion into a mammalian body cavity or openingdefined in a mammalian body, wherein the at least one first lightemitting element is supported by the probe.

In another aspects, devices and/or methods disclosed herein may be usedto modulate nitric oxide for managing or eliminating pathogens (such asbacteria, viruses, fungi, protists, or the like) in or on mammaliantissue. In additional aspects, devices and/or methods disclosed hereinmay be used to modulate nitric oxide for inhibiting 5α-reductase inmammalian tissue. In additional aspects, devices and/or methodsdisclosed herein may be used to modulate nitric oxide to promotecollagen synthesis. In additional aspects, devices and/or methodsdisclosed herein may be used to increase NO to levels suitable to inducecell death. In further aspects, devices and/or methods disclosed hereinmay be used for generation of, or promoting reaction with, reactiveoxygen species and free radicals. In additional aspects, devices and/ormethods disclosed herein may be used to increase vasodilation anddecrease inflammation.

In illustrative embodiments, provided is a method for treating aviral-infected tissue, the method comprises irradiating the tissue witha light from a light source with a particular dose (J/cm²), andrepeating the irradiating step for N iterations to constitute atreatment duration, wherein N is an integer greater than 1. In oneembodiment, the method comprises delivering a light dosage of at leastabout 10 J/cm² per day. In another embodiment, the method comprisesdelivering a light dosage of between about 10 to about 100 J/cm² perday. In certain examples, N is between 2 and 21 and the irradiating stepcould occur once, twice, or thrice a day. In some embodiments, N is 10or greater. As an example, the period of time could be for 1 to about 10minutes. In other embodiments, repeating occurs at least daily for atleast 3 days. In still other embodiments, the period of time is atgreater than 10 minutes, irradiating occurs at least twice daily for atleast 3 days.

In preferred embodiments, the light source, such as laser light, LEDlight, OLED light, and the like, any of which can be pulsed, is visiblelight ranging from 400 to 700 nm that provides minimal damage toepithelial tissue. In one illustrative embodiment, the light sourceincludes an LED with a spectral maximum between about 420 nm and about430 nm. In another embodiment, the light source or the light therefromis devoid of emissions of any wavelength conversion material stimulatedby the incoherent light emissions including a first peak wavelength in arange of from 410 nm to 440 nm. In another embodiment, the tissue isdevoid of an applied or received photosensitive therapeutic compound oragent. In another embodiment, at least 65% of a fluence of lightirradiating the tissue consists of the incoherent light emissionsincluding a first peak wavelength in a range of from 410 to 440 nm. Inanother embodiment, the light source or the light therefrom issubstantially devoid of light emissions having a peak wavelength in arange of from 441 nm to 490 nm.

Embodiments of antiviral phototherapy detailed in this disclosure can beeffective against both DNA and RNA virus infections. According to someembodiments, provided herein are methods of treating and/or preventing aviral infection. A method of treating and/or preventing a viralinfection may comprise administering light to the skin of a subject,thereby treating and/or preventing the viral infection in the subject.In some embodiments, a method may suppress and/or inhibit viralreplication of a virus and/or enhance the local immune response of asubject. In some embodiments, a method of treating and/or preventing avirus-related gastrointestinal condition may comprise administeringlight via colorectal administration via a probe inserted into the bodycavity of a subject, thereby treating and/or preventing thevirus-related colorectal or intestinal condition in the subject. Virusesin the GI tract include rotavirus, picornavirus, and coronavirus. Inother embodiments, a method of treating and/or preventing avirus-related central nervous system (CNS) infection may compriseadministering light transcranially, through the nose of a patient, orupon implantation of a light source into the tissue of a subject,thereby treating and/or preventing the virus-related CNS condition inthe subject. In specific embodiments, intranasal administration to thenasal mucosa can be used as a method of treating and/or preventing avirus-related infection. According to other embodiments, a method oftreating and/or preventing a virus-related bloodstream infection maycomprise transdermal administration of light to superficial vasculature,administering light to blood passed through an extra-corporeal loop,shining light on a blood product derived from the patient for use onother patients, and other methods for illumination of biological fluidsof a subject, thereby treating and/or preventing the virus-related bloodstream infection in the subject. In other embodiments, the light isapplied external to the body to the joints including those in the feetand hands, as well as the ankles, elbows, knees, and shoulders as amethod of treating and/or preventing a joint arthritis related to sideeffects caused by autoimmune reactions to viruses.

Further embodiments of the present disclosure describe an intravaginallight delivery device configured for delivering illumination totreatment areas in and around a cervix, the device comprising acylindrical shaft removably inserted within a flexible light coverhaving a light transmission portion, wherein, the cylindrical shaftcomprises a light source and control hardware therefore being orientedto transmit light in an axial direction from the cylindrical shaft, andthe flexible light cover is a hollow cylinder having an inside diametermatched to an outer diameter of the cylindrical shaft so that slidingthe flexible light cover over the cylindrical shaft nests thecylindrical shaft within so that the light source is positioned totransmit light through a light transmission portion.

In one embodiment, the light source comprises an LED with a spectralmaximum between about 420 nm and about 430 nm. In another embodiment,the light source is devoid of emissions of any wavelength conversionmaterial stimulated by the incoherent light emissions including a firstpeak wavelength in a range of from 410 nm to 440 nm. In one embodiment,the light source provides at least 65% of a fluence of light having afirst peak wavelength in a range of from 410 to 440 nm. In anotherembodiment, the light source is substantially devoid of light emissionshaving a peak wavelength in a range of from 441 nm to 490 nm. In yetanother embodiment, the light source delivers a radiant flux of 5 mW to60 mW. In other embodiments, the light source has at least one of thefollowing features: a light output of between 1 and 15 J cm⁻² min⁻¹, afirst peak wavelength between about 410 and 440 nm with a full widthhalf maximum (FWHM) of less than about 20 nm, is substantially devoid ofultraviolet radiation emissions, is substantially devoid of lightemissions having a peak wavelength in a range of from 441 to 490 nm, oris capable of delivering about 100 J cm⁻² in 10 minutes, 30 minutes, 1hour, or 4 hours. In illustrative embodiments, the intravaginal lightdelivery device further includes a battery or power supply capable ofdelivering about 100 J cm⁻² in 10 minutes, 30 minutes, 1 hour, or 4hours.

In illustrative embodiments, the intravaginal light delivery deviceincludes a flexible light cover with a treatment cup disposed about thelight transmission portion. In another embodiment, the flexible lightcover further comprises a cervical probe configured to spread cervicalsurfaces such that the cervical probe extends within the cervix, thecervical probe being configured to transmit light. In anotherembodiment, the flexible light cover includes a reversibly extendiblecup. In another embodiment, the treatment cup is asymmetric. As such,the treatment cup may be non-axially oriented to provide light deliveryat an angle of greater than about 5 degrees from an axis defined by thecenter of the cylindrical shaft.

In another aspect, a method comprises: providing a light sourceconfigured to emit light comprising a first light characteristic; andirradiating mammalian tissue within a body with the light to induce abiological effect, wherein the biological effect comprises altering aconcentration of one or more pathogens within the body and alteringgrowth of the one or more pathogens within the body. In certainembodiments, the first light characteristic comprises at least one of afirst peak wavelength and a radiant flux. In certain embodiments, thefirst light characteristic is the first peak wavelength and the firstpeak wavelength is in a range from 400 nanometers (nm) to 900 nm, or ina range from 400 nm to 450 nm, or in a range from 410 nm to 440 nm. Incertain embodiments, less than 5% of the light is in a wavelength rangethat is less than 400 nm. In certain embodiments, a full width halfmaximum of the first peak wavelength is less than or equal to 100 nm, orless than or equal to 40 nm. In certain embodiments, the first lightcharacteristic is the radiant flux and the radiant flux is in a rangefrom 5 milliwatts (mW) to 5000 mW. In certain embodiments, the radiantflux is configured to provide an irradiance to the tissue in a rangefrom 5 mW per square centimeter (mW/cm²) to 200 mW/cm².

In certain embodiments, the biological effect comprises inactivating theone or more pathogens that are in a cell-free environment in the bodyand inhibiting replication of the one or more pathogens that are in acell-associated environment in the body. In certain embodiments, thebiological effect further comprises upregulating a local immune responsewithin the body. In certain embodiments, the biological effect comprisesstimulating at least one of enzymatic generation of nitric oxide toincrease endogenous stores of nitric oxide and releasing nitric oxidefrom endogenous stores of nitric oxide.

In certain embodiments, impinging light to the tissue within the bodycomprises administering a dose of light in a range from 0.5 joules persquare centimeter (J/cm²) to 100 J/cm², or in a range from 2 J/cm² to 50J/cm². In certain embodiments, administering the dose of light comprisesproviding light with an irradiance to the tissue that is in a range from5 mW/cm² to 200 mW/cm² over a time period in a range from 10 seconds to1 hour. In certain embodiments, the irradiance is delivered in acontinuous manner. In certain embodiments, the irradiance is deliveredin a plurality of discrete pulses. In certain embodiments, the pluralityof discrete pulses comprises a plurality of equal pulses that isdelivered during the time period. In certain embodiments, the pluralityof discrete pulses comprises a plurality of dissimilar pulses that isdelivered during the time period In certain embodiments, the dose oflight is repeatably administered to provide a cumulative dose in a rangefrom 1 J/cm² to 1000 J/cm² over a cumulative time period. In certainembodiments, the dose of light is in a range from 0.5 J/cm² to 50 J/cm²and the dose of light is repeatably administered at least two times overthe cumulative time period. In certain embodiments, administering thedose of light comprises providing light with an irradiance in a rangefrom 0.1 mW/cm² to 10 watts per square centimeter (W/cm²) over a timeperiod in a range from 10 seconds to 1 hour, wherein the irradiance isdelivered in a plurality of discrete pulses.

In certain embodiments, impinging light to the tissue within the bodycomprises administering a dose of light with a light therapeutic indexof greater than or equal to 2, the light therapeutic index being definedas a dose concentration that reduces tissue viability by 25% divided bya dose concentration that reduces cellular percentage of the pathogensby 50%. In certain embodiments, light therapeutic index is in a rangefrom 2 to 250.

In certain embodiments, the one or more pathogens comprise at least oneof a virus, a bacteria, and a fungus. In certain embodiments, the one ormore pathogens comprise coronaviridae. In certain embodiments, the oneor more pathogens comprise orthomyxoviridae. In certain embodiments, oneor more pathogens comprise at least two types of viruses. In certainembodiments, the one or more pathogens comprise coronaviridae andorthomyxoviridae.

In certain embodiments, the light is provided by at least one of alight-emitting diode, an organic light-emitting diode, and a laser.

In certain embodiments, the tissue comprises mucosal epithelial tissue.In certain embodiments, the light is provided at a tissue depth of lessthan or equal to 2.5 mm.

In certain embodiments, the first light characteristic is a first peakwavelength and the first peak wavelength is in a range from 400 nm to900 nm and the light further comprises a second peak wavelength that isin a range from 400 nm to 900 nm, wherein the second peak wavelengthdiffers from the first peak wavelength by at least 10 nm. In certainembodiments, a full width half maximum of the second peak wavelength isless or equal to 100 nm. In certain embodiments, impinging light to thetissue within the body comprises administering first dose of light and asecond dose of light for a single type of microorganism.

In another aspect, a method comprises: providing light comprising afirst peak wavelength and a second peak wavelength; and irradiatingmammalian tissue with the light; wherein the first peak wavelengthdiffers from the second wavelength by at least 5 nm, the first peakwavelength is configured to induce a first biological effect, and thesecond peak wavelength is configured to induce a second biologicaleffect that is different than the first biological effect. In certainembodiments, the first biological effect and the second biologicaleffect comprise different ones of inactivating pathogens that are in acell-free environment, inhibiting replication of pathogens that are in acell-associated environment, upregulating a local immune response,stimulating enzymatic generation of nitric oxide to increase endogenousstores of nitric oxide, releasing nitric oxide from endogenous stores ofnitric oxide, and inducing an anti-inflammatory effect. In certainembodiments, the first peak wavelength is in a range from 400 nm to 900nm and the second peak wavelength is in a range from 400 nm to 900 nm.In certain embodiments, the first peak wavelength is in a range from 400nm to 490 nm and the second peak wavelength is in a range from 490 nm to900 nm. In certain embodiments, the first peak wavelength is in a rangefrom 400 nm to 490 nm and the second peak wavelength is in a range from320 nm to 400 nm. In certain embodiments, the first peak wavelength is arange of from 410 nm to 440 nm. In certain embodiments, the lightfurther comprises a third peak wavelength that is configured to induce athird biological effect that is different than the first biologicaleffect and the second biological effect, wherein: the first peakwavelength is in a range from 400 nm to 490 nm; the second peakwavelength is in a range from 490 nm to 900 nm; and the third peakwavelength is in a range from 200 nm to 400 nm.

In certain embodiments, impinging light to the tissue comprisesadministering the first peak wavelength in a first time window and thesecond peak wavelength in a second time window. In certain embodiments,the first time window is the same as the second time window. In certainembodiments, the first time window is different than the second timewindow. In certain embodiments, the first time window overlaps with thesecond time window. In certain embodiments, the first time window isnon-overlapping with the second time window.

In another aspect, a method comprises: providing a first dose of lightto mammalian tissue that is configured to induce a first biologicaleffect for a first pathogen; and providing a second dose of light to themammalian tissue that is configured to induce a second biological effectfor at least one of the first pathogen and a second pathogen, whereinthe first pathogen is different than the second pathogen. In certainembodiments, the first biological effect comprises at least one ofinactivating the first pathogen in a cell-free environment, inhibitingreplication of the first pathogen in a cell-associated environment,upregulating a local immune response in the mammalian tissue,stimulating enzymatic generation of nitric oxide to increase endogenousstores of nitric oxide in the mammalian tissue, releasing nitric oxidefrom endogenous stores of nitric oxide in the mammalian tissue, andinducing an anti-inflammatory effect in the mammalian tissue. In certainembodiments, the second biological effect comprises at least one ofinactivating the second pathogen in a cell-free environment, inhibitingreplication of the second pathogen in a cell-associated environment,upregulating a local immune response in the mammalian tissue,stimulating enzymatic generation of nitric oxide to increase endogenousstores of nitric oxide in the mammalian tissue, releasing nitric oxidefrom endogenous stores of nitric oxide in the mammalian tissue, andinducing an anti-inflammatory effect in the mammalian tissue. In certainembodiments, the first pathogen comprises at least one of a virus, abacteria, and a fungus and the second pathogen comprises a different oneof a virus, a bacteria, and a fungus.

In certain embodiments, the first dose of light is administered with afirst light therapeutic index of greater than or equal to 2, the firstlight therapeutic index being defined as a dose concentration of thefirst dose that reduces viability of the mammalian tissue by 25% dividedby a dose concentration of the first dose that reduces cellularpercentage of the first pathogen by 50%; and the second dose of light isadministered with a second light therapeutic index of greater than orequal to 2, the second light therapeutic index being defined as a doseconcentration of the second dose that reduces viability of the mammaliantissue by 25% divided by a dose concentration of the second dose thatreduces cellular percentage of the second pathogen by 50%. In certainembodiments, the first light therapeutic index and the second lighttherapeutic index are both in a range from 2 to 250.

In certain embodiments, the first dose of light comprises a first peakwavelength in a range from 400 nm to 490 nm, and the second dose oflight comprises a second peak wavelength in a range from 490 nm to 900nm. In certain embodiments, the first dose of light comprises a firstpeak wavelength in a range from 400 nm to 490 nm, and the second dose oflight comprises a second peak wavelength in a range from 320 nm to 400nm. In certain embodiments, each of the first dose of light and thesecond dose of light are in a range from 0.5 J/cm² to 100 J/cm². Incertain embodiments, the first dose of light and the second dose oflight are repeatably administered to provide a cumulative dose in arange from 1 J/cm² to 1000 J/cm². In certain embodiments, the first doseof light comprises a first peak wavelength in a range from 410 nm to 440nm.

In another aspect, an illumination device comprises: at least one lightsource arranged to impinge light on mammalian tissue within a body, thelight comprising a first light characteristic and configured to induce abiological effect; and driver circuitry configured to drive the at leastone light source; wherein the biological effect comprises altering aconcentration of one or more pathogens within the body and alteringgrowth of the one or more pathogens within the body. In certainembodiments, the illumination device further comprises an optic that isarranged to pass the light from the at least one light source forirradiating the mammalian tissue within the body. In certainembodiments, the optic is further arranged in optical communication witha camera for viewing the mammalian tissue within the body. In certainembodiments, the optic resides on an illumination head of theillumination device and the illumination head is angled from alengthwise direction of the illumination device. In certain embodiments,the illumination head is removably attached to the illumination device.In certain embodiments, the illumination device further comprises alight guide that is arranged between the optic and the at least on lightsource. In certain embodiments, the illumination device furthercomprises a protective covering that comprises a same material as theoptic. In certain embodiments, the illumination device is configured tobe user controlled.

In certain embodiments, the illumination device is configured to be atleast partially inserted within a body cavity, wherein the mammaliantissue is included within the body cavity. In certain embodiments, theat least one light source is arranged outside of the body cavity whenthe illumination device is partially inserted within the body cavity. Incertain embodiments, the at least one light source is arranged withinthe body cavity when the illumination device is partially insertedwithin the body cavity. In certain embodiments, the illumination deviceis configured to be fully inserted within a body cavity, wherein themammalian tissue is included within the body cavity. In certainembodiments, the illumination device further comprises a cable that isconfigured to retrieve the illumination device from the body cavity.

In certain embodiments, the illumination device further comprises amicrocontroller that is configured to control the driver circuitry. Incertain embodiments, the microcontroller is further configured toreceive an input from at least one sensor for controlling the at leastone light source. In certain embodiments, the at least one sensorcomprises one or more of a temperature sensor and a proximity sensor.

In certain embodiments, the biological effect further comprisesupregulating a local immune response within the body. In certainembodiments, the biological effect comprises stimulating at least one ofenzymatic generation of nitric oxide to increase endogenous stores ofnitric oxide and releasing nitric oxide from endogenous stores of nitricoxide. In certain embodiments, the biological effect comprisesinactivating the one or more pathogens that are in a cell-freeenvironment within the body. In certain embodiments, the biologicaleffect comprises inhibiting replication of the one or more pathogensthat are in a cell-associated environment within the body.

In certain embodiments, impinging the light on the mammalian tissuewithin the body comprises administering a dose of light in a range from0.5 J/cm² to 100 J/cm². In certain embodiments, the first lightcharacteristic comprises at least one of a first peak wavelength and aradiant flux. In certain embodiments, the first light characteristic isthe first peak wavelength and the first peak wavelength is in a rangefrom nm to 900 nm. In certain embodiments, the first peak wavelength isin a range from 410 nm to 440 nm. In certain embodiments, the lightfurther comprises a second peak wavelength in a range from 400 nm to 900nm, and the second peak wavelength is different than the first peakwavelength.

In another aspect, a method comprises: providing light comprising afirst peak wavelength in a range from 400 nm to 900 nm; andadministering a dose of the light to mammalian tissue within a body toinduce a biological effect, the dose of light comprising providing anirradiance to the mammalian tissue over a time period of at most 1 hour,the irradiance being delivered in a plurality of discrete pulses;wherein the biological effect comprises altering a concentration of oneor more pathogens within the body and altering growth of the one or morepathogens within the body. In certain embodiments, the irradiance is arange from 0.1 mW/cm² to 10 W/cm². In certain embodiments, the dose oflight is a range from 0.5 (J/cm² to 100 J/cm². In certain embodiments,the dose of light is in a range from 2 J/cm² to 50 J/cm². In certainembodiments, the plurality of discrete pulses comprises a plurality ofequal pulses that is delivered during the time period. In certainembodiments, the plurality of discrete pulses comprises a plurality ofdissimilar pulses that is delivered during the time period. In certainembodiments, the irradiance progressively increases during the pluralityof dissimilar pulses. In certain embodiments, the irradianceprogressively decreases during the plurality of dissimilar pulses. Incertain embodiments, the dose of light is repeatably administered toprovide a cumulative dose in a range from one J/cm² to 1000 J/cm² over acumulative time period. In certain embodiments, the dose of light isprovided with a light therapeutic index of greater than or equal to 2,the light therapeutic index being defined as a dose concentration thatreduces tissue viability by 25% divided by a dose concentration thatreduces cellular percentage of the one or more pathogens by 50%.

In another aspect, any of the foregoing aspects, and/or various separateaspects and features as described herein, may be combined for additionaladvantage. Any of the various features and elements as disclosed hereinmay be combined with one or more other disclosed features and elementsunless indicated to the contrary herein.

Those skilled in the art will appreciate the scope of the presentdisclosure and realize additional aspects thereof after reading thefollowing detailed description of the preferred embodiments inassociation with the accompanying drawing figures.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawing figures incorporated in and forming a part ofthis specification illustrate several aspects of the disclosure, andtogether with the description serve to explain the principles of thedisclosure.

FIG. 1 is a reaction sequence showing photoactivated production ofnitric oxide (NO) catalyzed by iNOS, followed by binding of NO to CCO.

FIG. 2A is a reaction sequence showing photomodulated release of NO fromnitrosothiols (RSNO).

FIG. 2B is a reaction sequence showing photomodulated release of NO frommetal nitrosyls (M-NO).

FIG. 2C is a reaction sequence showing loading of cytochrome c oxidase(CCO) with NO (yielding CCO-NO and CCO-NO₂ ⁻) followed by photomodulatedrelease of nitric oxide from the CCO-NO and CCO-NO₂ ⁻.

FIG. 3 is a cross-sectional view of epidermis and dermis layers of humanskin with schematic illustration of overlapping zones in which NO isreleased from endogenous stores of NO by photomodulation.

FIG. 4A includes superimposed plots of absorbance versus wavelength forhemoglobin (Hb), nitric oxide-loaded hemoglobin (Hb-NO) prior toirradiation, and Hb-NO following absorption of 150 J of light emissionsof a green LED having a peak wavelength of 530 nm.

FIG. 4B includes superimposed plots of absorbance versus wavelength forHb, Hb-NO prior to irradiation, and Hb-NO following absorption of 150 Jof light emissions of an IR LED source having a peak wavelength of 850nm.

FIG. 5 is a plot of percentage change in peak absorbance for the 540 nmpeak of Hb-NO versus fluence (Joules per square centimeter) for ninedifferent wavelengths of light (from 410 nm to 850 nm).

FIG. 6 is a plot of percentage change in peak absorbance for Cytochromec-NO versus fluence (Joules per square centimeter) for nine differentwavelengths of light (from 410 nm to 850 nm).

FIG. 7 is a plot of released NO (ppb) per milliwatt per squarecentimeter versus time for the photomodulated release of NO from Hb-NOfor nine different wavelengths of light (from 410 nm to 850 nm).

FIG. 8A includes superimposed plots of released NO (ppb) per milliwattper square centimeter versus time for irradiation of Hb-NO with (i) a410 nm peak wavelength blue LED device, (ii) a 530 nm peak wavelengthgreen LED device, and (iii) the 410 nm peak wavelength blue LED devicein combination with the 530 nm peak wavelength green LED device.

FIG. 8B includes superimposed plots of released NO (ppb) per milliwattper square centimeter versus time for irradiation of Hb-NO with (i) a530 nm peak wavelength green LED device, (ii) a 660 nm peak wavelengthred LED device, and (iii) the 530 nm peak wavelength green LED device incombination with the 660 nm peak wavelength red LED device.

FIG. 8C includes superimposed plots of released NO (ppb) per milliwattper square centimeter versus time for irradiation of Hb-NO with (i) a530 nm peak wavelength green LED device (including one repeat run), (ii)a 850 nm peak wavelength infrared LED device (including one repeat run),and (iii) the 530 nm peak wavelength green LED device in combinationwith the 850 nm peak wavelength infrared LED device.

FIG. 9 is a side cross-sectional schematic view of a portion of a devicefor delivering light energy to living mammalian tissue, the deviceincluding multiple direct view light emitting sources supported by asubstrate and covered with an encapsulant material layer.

FIG. 10 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, thedevice including multiple direct view light emitting sources supportedby a substrate and covered with an encapsulant material layer, whereinat least one functional material (e.g., wavelength conversion and/orscattering material) is disposed within the encapsulant material layer.

FIG. 11 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, thedevice including multiple direct view light emitting sources supportedby a substrate and covered with two encapsulant material layers, with atleast one functional material (e.g., wavelength conversion and/orscattering material) layer disposed between the encapsulant materiallayers.

FIG. 12 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, thedevice including multiple direct view light emitting sources supportedby a substrate and covered by an encapsulant layer, wherein theencapsulant layer is covered with a diffusion or scattering materiallayer.

FIG. 13 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, thedevice including multiple direct view light emitting sources supportedby a substrate, multiple molded features overlying the light emittingsources, and an encapsulant or light coupling material arranged betweenthe light emitting sources and the molded features.

FIG. 14 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, thedevice including a flexible substrate, one or more organic lightemitting diode layers arranged between an anode and cathode, and anencapsulant layer arranged over the cathode.

FIG. 15 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, thedevice including a flexible substrate, multiple direct view lightemitting sources supported by the substrate, encapsulant material layersarranged above and below the substrate and over the light emittingsources, and holes or perforations defined through both the substrateand the encapsulant material layers.

FIG. 16 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device includes multiple direct view light emitting sourcessupported by a substrate and covered by an encapsulant layer, and thedevice is arranged in a concave configuration.

FIG. 17 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device includes multiple direct view light emitting sourcessupported by a substrate and covered by an encapsulant layer, and thedevice is arranged in a convex configuration.

FIG. 18 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device is edge lit with one or more light emitting sources supportedby a flexible printed circuit board (PCB), other non-light-transmittingsurfaces of the device are bounded by a flexible reflective substrate,and the flexible PCB and light emitting source(s) are covered with anencapsulant material.

FIG. 19 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device is edge lit with one or more light emitting sources supportedby a flexible printed circuit board (PCB), anothernon-light-transmitting surface of the device is bounded by a flexiblereflective substrate, the flexible PCB and light emitting source(s) arecovered with an encapsulant material, and the device is tapered inthickness.

FIG. 20 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device is edge lit with one or more light emitting sources supportedby a flexible PCB having a reflective surface, non-light-transmittingsurfaces of the device are further bounded by the flexible PCB, and theflexible PCB and light emitting source(s) are covered with anencapsulant material.

FIG. 21 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device is edge lit with one or more light emitting sources supportedby a flexible PCB having a reflective surface, anothernon-light-transmitting surface of the device is further bounded by theflexible PCB, the flexible PCB and light emitting source(s) are coveredwith an encapsulant material, and the device is tapered in thickness.

FIG. 22 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device is edge lit with one or more light emitting sources supportedby a flexible PCB having a reflective surface, othernon-light-transmitting surfaces of the device are further bounded by theflexible PCB, the flexible PCB and light emitting source(s) are coveredwith an encapsulant material, and a light-transmitting face of thedevice includes a diffusing and/or scattering layer.

FIG. 23 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device is edge lit with one or more light emitting sources supportedby a flexible PCB having a reflective surface, anothernon-light-transmitting surface of the device is further bounded by theflexible PCB, the flexible PCB and light emitting source(s) are coveredwith an encapsulant material, a light transmitting face of the device istapered in thickness, and the light-transmitting face includes adiffusing and/or scattering layer.

FIG. 24 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device is edge lit with one or more light emitting sources supportedby a flexible PCB having a reflective surface, othernon-light-transmitting surfaces of the device are further bounded by theflexible PCB, the flexible PCB and light emitting source(s) are coveredwith an encapsulant material, and a light-transmitting face of thedevice includes a wavelength conversion material layer.

FIG. 25 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device is edge lit with one or more light emitting sources supportedby a flexible PCB having a reflective surface, anothernon-light-transmitting surface of the device is further bounded by theflexible PCB, the flexible PCB and light emitting source(s) are coveredwith an encapsulant material, a light transmitting face of the device istapered in thickness, and the light-transmitting face includes awavelength conversion material layer.

FIG. 26 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device is edge lit along multiple edges with multiple light emittingsources supported by a flexible PCB having a reflective surface, othernon-light-transmitting surfaces of the device are further bounded by theflexible PCB, the flexible PCB and light emitting sources are coveredwith an encapsulant material, and a wavelength conversion material isdistributed in the encapsulant material.

FIG. 27 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device is edge lit along multiple edges with multiple light emittingsources supported by a flexible PCB having a reflective surface, othernon-light-transmitting surfaces of the device are further bounded by theflexible PCB with raised light extraction features being supported bythe flexible PCB, and encapsulant material is provided over the flexiblePCB, the light emitting sources, and the light extraction features.

FIG. 28 is a side cross-sectional schematic view of a portion of adevice for delivering light energy to living mammalian tissue, whereinthe device is edge lit along multiple edges with multiple light emittingsources supported by a flexible PCB having a reflective surface, othernon-light-transmitting surfaces of the device are further bounded by theflexible PCB, an encapsulant material is arranged above and below thePCB and over the light emitting sources, and holes or perforations aredefined through both the substrate and the encapsulant material.

FIG. 29A is a cross-sectional view of a first exemplary hole definablethrough a device for delivering light energy to living mammalian tissue,the hole having a diameter that is substantially constant with depth.

FIG. 29B is a cross-sectional view of a second exemplary hole definablethrough a device for delivering light energy to living mammalian tissue,the hole having a diameter that increases with increasing depth.

FIG. 29C is a cross-sectional view of a third exemplary hole definablethrough a device for delivering light energy to living mammalian tissue,the hole having a diameter that decreases with increasing depth.

FIG. 30 is a top schematic view of at least a portion of a device fordelivering light energy to living mammalian tissue, wherein the deviceis edge lit along multiple edges with multiple light emitting sourcessupported by a flexible PCB, and multiple holes or perforations ofsubstantially uniform size and substantially uniform distribution aredefined through the flexible PCB.

FIG. 31 is a top schematic view of at least a portion of a device fordelivering light energy to living mammalian tissue, wherein the deviceis edge lit along multiple edges with multiple light emitting sourcessupported by a flexible PCB, and multiple holes or perforations ofdifferent sizes but with a substantially uniform distribution aredefined through the flexible PCB.

FIG. 32 is a top schematic view of at least a portion of a device fordelivering light energy to living mammalian tissue, wherein the deviceis edge lit along multiple edges with multiple light emitting sourcessupported by a flexible PCB, and multiple holes or perforations ofdifferent sizes are provided in clusters and defined through theflexible PCB proximate to selected light emitting sources.

FIG. 33 is a top schematic view of at least a portion of a device fordelivering light energy to living mammalian tissue, wherein the deviceis edge lit along multiple edges with multiple light emitting sourcessupported by a flexible PCB, and multiple holes or perforations ofdifferent sizes and with a non-uniform (e.g., random) distribution aredefined through the flexible PCB.

FIG. 34A is a top schematic view of at least a portion of a lightemitting device for delivering light energy to living mammalian tissueand at least a portion of a battery/ control module, wherein anelongated electrical cord is associated with the battery/ control modulefor connecting the battery/control module to the light emitting device.

FIG. 34B is a top schematic view of at least a portion of a lightemitting device for delivering light energy to living mammalian tissueand at least a portion of a battery/ control module, wherein anelongated electrical cord is associated with the light emitting devicefor connecting the light emitting device to the battery/control module.

FIG. 35 is a top schematic view of at least a portion of a lightemitting device for delivering light energy to living mammalian tissueand being connected via an electrical cord to a battery/control module,wherein the light emitting device includes multiple light emitters,multiple holes or perforations, and multiple sensors.

FIG. 36A is a plot of intensity versus time (t) embodying a firstexemplary illumination cycle that may be used with at least one emitterof a light emitting device for delivering light energy to livingmammalian tissue as disclosed herein.

FIG. 36B is a plot of intensity versus time (t) embodying a secondexemplary illumination cycle that may be used with at least one emitterof a light emitting device for delivering light energy to livingmammalian tissue as disclosed herein.

FIG. 36C is a plot of intensity versus time (t) embodying a thirdexemplary illumination cycle that may be used with at least one emitterof a light emitting device for delivering light energy to livingmammalian tissue as disclosed herein.

FIG. 37 is an exploded view of a light emitting device embodied in awearable cap for delivering light energy to a scalp of a patient, thedevice including at least one light emitter supported by a flexible PCBarranged in a concave configuration, a concave support member shaped toreceive the flexible PCB and support a battery and control module, and afabric covering arranged to cover the support member and flexiblesubstrate.

FIG. 38 is a bottom plan view of the flexible PCB of FIG. 37 prior tobeing shaped into a concave configuration.

FIG. 39 is a front elevation view of the light emitting device of FIG.37 affixed to a modeled human head.

FIG. 40 is a schematic diagram showing interconnections betweencomponents of a light emitting device or delivering light energy totissue of a patient according to one embodiment.

FIG. 41 is a schematic diagram depicting an interface between hardwaredrivers, functional components, and a software application suitable foroperating a light emitting device according to FIG. 40.

FIG. 42 is a schematic elevation view of at least a portion of a lightemitting device for delivering light energy to tissue in an internalcavity of a patient according to one embodiment.

FIG. 43A is a schematic elevation view of at least a portion of a lightemitting device including a concave light emitting surface fordelivering light energy to cervical tissue of a patient according to oneembodiment.

FIG. 43B illustrates the device of FIG. 43A inserted into a vaginalcavity to deliver light energy to cervical tissue of a patient.

FIG. 44A is a schematic elevation view of at least a portion of a lightemitting device including a probe-defining light emitting surface fordelivering light energy to cervical tissue of a patient according toanother embodiment.

FIG. 44B illustrates the device of FIG. 44A inserted into a vaginalcavity, with a probe portion of the light-emitting surface inserted intoa cervical opening, to deliver light energy to cervical tissue of apatient.

FIG. 45 is a bar chart identifying percentage of viable cells as afunction of time post 420 nm irradiation (from 0 to 24 hours) for fourdifferent fluence values ranging from 0 J/cm² to 50 J/cm² for NOgeneration in keratinocytes resulting from photobiomodulation.

FIG. 46 is a bar chart identifying percentage of cells expressing iNOSas a function of time post 420 nm irradiation (from 0 to 8 hours) forfour different fluence values ranging from 0 J/cm² to 50 J/cm² for NOgeneration in keratinocytes resulting from photobiomodulation.

FIG. 47 is a bar chart identifying percentage of cells expressing nNOSas a function of time post 420 nm irradiation (from 0 to 8 hours) forfour different fluence values ranging from 0 J/cm² to 50 J/cm² for NOgeneration in keratinocytes resulting from photobiomodulation.

FIG. 48 is a bar chart identifying percentage of cells withintracellular NO as a function of time post 420 nm irradiation (from 0to 8 hours) for four different fluence values ranging from 0 J/cm² to 50J/cm² for NO generation in keratinocytes resulting fromphotobiomodulation.

FIG. 49 is a bar chart identifying percentage of viable cells as afunction of time post 420 nm irradiation (from 0 to 24 hours) for fourdifferent fluence values ranging from 0 J/cm² to 50 J/cm² for NOgeneration in fibroblasts resulting from photobiomodulation.

FIG. 50 is a bar chart identifying percentage of cells expressing iNOSas a function of time post 420 nm irradiation (from 0 to 6 hours) forfour different fluence values ranging from 0 J/cm² to 50 J/cm² for NOgeneration in fibroblasts resulting from photobiomodulation.

FIG. 51 is a bar chart identifying percentage of cells expressing eNOSas a function of time post 420 nm irradiation (from 0 to 6 hours) forfour different fluence values ranging from 0 J/cm² to 50 J/cm² for NOgeneration in fibroblasts resulting from photobiomodulation.

FIG. 52 is a bar chart identifying percentage of cells withintracellular NO as a function of time post 420 nm irradiation (from 0to 6 hours) for four different fluence values ranging from 0 J/cm² to 50J/cm² for NO generation in fibroblasts resulting fromphotobiomodulation.

FIG. 53 is a plot of NO release rate (PPB/s) versus irradiance (J/cm²)from hemoglobin-NO for nine (9) different wavelengths of incoherentlight ranging from 410 nm to 850 nm.

FIG. 54 is a plot of total NO release (PPB) versus irradiance (J/cm²)from hemoglobin-NO for nine (9) different wavelengths of incoherentlight ranging from 410 nm to 850 nm.

FIG. 55 is a plot of NO release rate (PPB/s) versus irradiance (J/cm²)from S-nitrosoglutathione (GSNO) for ten (10) different wavelengths ofincoherent light ranging from 410 nm to 850 nm.

FIG. 56 is a plot of total NO release (PPB) versus irradiance (J/cm²)from S-nitrosoglutathione (GSNO) for ten (10) different wavelengths ofincoherent light ranging from 410 nm to 850 nm.

FIG. 57 is a plot of NO release rate (PPB/s) versus irradiance (J/cm²)from albumin-NO for nine (9) different wavelengths of incoherent lightranging from 420 nm to 850 nm.

FIG. 58 is a plot of total NO release (PPB) versus irradiance (J/cm²)from albumin-NO for nine (9) different wavelengths of incoherent lightranging from 420 nm to 850 nm.

FIG. 59 is a plot of NO release rate (PPB/s) versus irradiance (J/cm²)from cytochrome c-NO for ten (10) different wavelengths of incoherentlight ranging from 410 nm to 850 nm.

FIG. 60 is a plot of total NO release (PPB) versus irradiance (J/cm²)from cytochrome c-NO for ten (10) different wavelengths of incoherentlight ranging from 410 nm to 850 nm.

FIG. 61 is a plot of NO release rate (PPB/s) versus irradiance (J/cm²)from cytochrome c-oxidase-NO for ten (10) different wavelengths ofincoherent light ranging from 410 nm to 850 nm.

FIG. 62 is a plot of total NO release (PPB) versus irradiance (J/cm²)from cytochrome c-oxidase-NO for ten (10) different wavelengths ofincoherent light ranging from 410 nm to 850 nm.

FIG. 63 is a plot of NO release rate (PPB/s) versus irradiance (J/cm²)from mitochondria-NO for ten (10) different wavelengths of incoherentlight ranging from 410 nm to 850 nm.

FIG. 64 is a plot of total NO release (PPB) versus irradiance (J/cm²)from mitochondria-NO for ten (10) different wavelengths of incoherentlight ranging from 410 nm to 850 nm.

FIG. 65 is a related art perspective view illustration of across-section of dermis and epidermis layers of human skin showingvarious types of cells containing nitric oxide compounds.

FIG. 66 is a related art cross-sectional illustration of human skin witha superimposed representation of depth penetration of coherent light ofeight different wavelengths ranging from 420 nm to 755 nm.

FIG. 67A is an upper perspective view photograph comparing thetransmittance of red (660 nm peak wavelength) incoherent (LED) light anda red (660 nm) coherent (laser) light through a human skin sample.

FIG. 67B is a plot of light transmittance percentage as a function ofskin thickness (mm) for transmittance of red (660 nm peak wavelength)incoherent (LED) light and a red (660 nm) coherent (laser) light throughhuman skin samples of two different thicknesses at equivalentirradiance.

FIG. 68A is an upper perspective view photograph comparing thetransmittance of a green (530 nm peak wavelength) incoherent (LED) lightand a green (530 nm) coherent (laser) light through a human skin sample.

FIG. 68B is a plot of light transmittance percentage as a function ofskin thickness (mm) for transmittance of green (530 nm peak wavelength)incoherent (LED) light and a green (530 nm) coherent (laser) lightthrough human skin samples of two different thicknesses at equivalentirradiance.

FIG. 69A is an upper perspective view photograph comparing thetransmittance of a blue (420 nm peak wavelength) incoherent (LED) lightand a blue (420 nm) coherent (laser) light through a human skin sample.

FIG. 69B is a plot of light transmittance percentage as a function ofskin thickness (mm) for transmittance of blue (420 nm peak wavelength)incoherent (LED) light and a blue (420 nm) coherent (laser) lightthrough human skin samples of two different thicknesses at equivalentirradiance.

FIG. 70 is a plot of light transmittance percentage as a function ofskin thickness (mm) for transmittance of red (660 nm peak wavelength)incoherent (LED) light and red (660 nm) coherent (laser) light throughhuman skin samples of two different pigmentations and three differentthicknesses.

FIG. 71 is a plot of light transmittance percentage as a function ofskin thickness (mm) for transmittance of green (530 nm peak wavelength)incoherent (LED) light and green (530 nm) coherent (laser) light throughhuman skin samples of two different pigmentations and three differentthicknesses.

FIG. 72 is a plot of light transmittance percentage as a function ofskin thickness (mm) for transmittance of blue (420 nm peak wavelength)incoherent (LED) light and blue (420 nm) coherent (laser) light throughhuman skin samples of two different pigmentations and three differentthicknesses.

FIG. 73 is a plot of percentage of DHT remaining as a function ofNO-donor concentration (mM) for six values ranging from 0 to 50 mM,showing that lower percentages of DHT remaining are correlated withincreased NO-donor concentrations.

FIG. 74 is a plot of percentage of DHT remaining as a function ofNO-donor concentration (mM) for dark conditions and 420 nm lightexposure conditions for NO-donor concentrations of 0 and 1 mM.

FIG. 75 is a diagrammatic drawing showing two potential mechanisms ofaction by which phototherapy may inhibit or eradicate viral infections.

FIG. 76 is a schematic illustration of the female reproductive system.

FIG. 77A is a schematic illustration of the inside of one embodiment ofan illumination device for delivering light energy to tissue.

FIG. 77B is a schematic illustration of the device shown in FIG. 77A.

FIG. 78A is a schematic illustration of another device for deliveringlight energy to tissue.

FIG. 78B is a schematic illustration of another device for deliveringlight energy to tissue.

FIGS. 79A-79F is an illustration of an experimental design for treatmentof human papillomavirus (HPV)-infected tissues where an HPV-infectedorganotypic epithelial raft culture model was used to prepareHPV-infected tissue for performing anti-viral experiments.

FIGS. 80A-80J are photomicrographs of organotypic epithelial cultureswhich were either healthy (FIGS. 80A-80E) or infected with HPV-18 (FIGS.80F-80J) where certain cultures were exposed to phototherapy over a10-minute time period.

FIGS. 81A-81J are additional photomicrographs of organotypic epithelialcultures which were either healthy (FIGS. 81A-81E) or infected withHPV-18 (FIGS. 81F-81J).

FIG. 82A is a chart illustrating measured spectral flux relative towavelength for different exemplary LED arrays.

FIG. 82B illustrates a perspective view of a testing set-up forproviding light from one or more LED arrays to a biological testarticle.

FIG. 83A is a chart illustrating a percent viability for a peakwavelength of 385 nm for a range of doses.

FIG. 83B is a chart illustrating a percent viability for a peakwavelength of 405 nm for the same doses of FIG. 83A.

FIG. 83C is a chart illustrating a percent viability for a peakwavelength of 425 nm for the same doses of FIG. 83A.

FIG. 84A is a chart illustrating percent viability for Vero E6 cells forantiviral assays performed on ninety-six well plates at various cellseeding densities.

FIG. 84B is a chart illustrating percent viability for Vero E6 cells forantiviral assays performed on forty-eight well plates at various cellseeding densities.

FIG. 84C is a chart illustrating percent viability for Vero E6 cells forantiviral assays performed on twenty-four well plates at various cellseeding densities.

FIG. 85A is a chart illustrating tissue culture infectious dose (TCID₅₀)per milliliter (ml) for the 425 nm light at the various dose ranges forVero E6 cells infected with a multiplicity of infection (MOI) of 0.001SARS-CoV-2 isolate USA-WA1/2020 for 1 hour.

FIG. 85B is a chart illustrating percent reduction in SARS-CoV-2replication versus percent cell cytotoxicity for the doses of light asillustrated in FIG. 62A.

FIG. 86A is a chart illustrating TCID₅₀/ml for 425 nm light at variousdose ranges for Vero E6 cells infected with a MOI of 0.01 SARS-CoV-2isolate USA-WA1/2020 for 1 hour.

FIG. 86B is a chart illustrating percent reduction in SARS-CoV-2replication versus percent cell cytotoxicity for the doses of light asillustrated in FIG. 63A.

FIG. 86C is a table showing an evaluation of SARS-CoV-2 RNA with reversetranscription polymerase chain reaction (rRT-PCR) for samples collectedfor the TCID₅₀ assays of FIGS. 86A-86B.

FIG. 87A is a chart illustrating TCID₅₀/ml for 425 nm light at variousdose ranges for Vero 76 cells infected with a MOI of 0.01 SARS-CoV-2.

FIG. 87B is a chart illustrating percent reduction in SARS-CoV-2replication versus percent cell cytotoxicity for the doses of light asillustrated in FIG. 87A.

FIG. 88 is a chart illustrating TCID₅₀/ml versus various doses of 625 nmred light for Vero E6 cells infected with a MOI of 0.01.

FIG. 89A is a chart illustrating a virus assay by TCID₅₀ on Vero E6cells for SARS-CoV-2 from a first laboratory.

FIG. 89B is a chart illustrating a virus assay by TCID₅₀ on Vero E6cells for SARS-CoV-2 from a first laboratory.

FIG. 90A is a chart indicating that Vero E6 cells do not show decreasedviability under 530 nm light at doses ranging from 0-180 J/cm².

FIG. 90B is a chart indicating that Vero E6 cells do not show decreasedviability under 625 nm light at doses ranging from 0-240 J/cm².

FIG. 91A is a chart showing raw luminescence values (RLU) for differentseedings of Vero E6 cell densities and various doses of light (J/cm²).

FIG. 91B is a chart showing percent viability for the different seedingsof Vero E6 cell densities and various doses of light of FIG. 91A.

FIG. 91C is a chart comparing RLU versus total cell number to show thatCellTiterGlo One Solution (CTG) is an effective reagent for measuringcell densities of above 10⁶ Vero E6 cells.

FIG. 92A is a chart of TCID₅₀/ml versus dose at 24 hours and 48 hourspost infection for Calu-3 cells infected with SARS-CoV-2.

FIG. 92B is a chart showing the percent reduction in SARS-Cov-2 comparedwith percent cytotoxicity for the Calu-3 cells of FIG. 92A.

FIG. 93A is a chart illustrating percent reduction in SARS-CoV-2replication versus percent cell cytotoxicity for Vero E6 cells infectedwith a MOI of 0.01 after various doses of light at 425 nm.

FIG. 93B is a chart illustrating percent reduction in SARS-CoV-2replication versus percent cell cytotoxicity for Vero E6 cells infectedwith a MOI of 0.001 after various doses of light at 425 nm.

FIG. 93C is a chart representing percent viability at various doses forprimary human tracheal/bronchial tissue from a single donor aftervarious doses of light at 425 nm.

FIG. 94A is a chart illustrating percent reduction in SARS-CoV-2replication versus percent cell cytotoxicity for Vero E6 cells infectedwith a MOI of 0.01 after various doses of light at 450 nm.

FIG. 94B is a chart illustrating percent reduction in SARS-CoV-2replication versus percent cell cytotoxicity for Vero E6 cells infectedwith a MOI of 0.001 after various doses of light at 450 nm.

FIG. 94C is a chart representing percent viability at various doses forprimary human tracheal/bronchial tissue from a single donor aftervarious doses of light at 450 nm.

FIG. 95 is a table summarizing the results illustrated in FIGS. 93A-93Cand 94A-94C.

FIG. 96A is a chart showing the titer of wild-type (WT) influenza Avirus based on remaining viral loads for different initial viral dosesafter treatment with different doses of 425 nm light.

FIG. 96B is a chart showing the titer of Tamiflu-resistant influenza Avirus based on remaining viral load for a single initial viral doseafter treatment of different doses of 425 nm light.

FIG. 97A is a chart showing the TCID₅₀/ml versus energy dose for WTinfluenza A treated with light at 425 nm at various doses with a MOI forthe WT influenza A of 0.01.

FIG. 97B is a plot showing the percent reduction in viral loads of WTinfluenza A and percent cytotoxicity against the treated cells wheninfluenza A-infected Madin-Darby Canine Kidney (MDCK) cells were exposedto 425 nm light at various doses and a MOI for the WT influenza A wasprovided at 0.01.

FIG. 97C illustrates the TCID₅₀ of cells infected with WT influenza Aand treated with 425 nm light at various doses with a MOI for the WTinfluenza A of 0.1.

FIG. 97D illustrates the percent reduction in viral loads of WTinfluenza A and percent cytotoxicity against the treated cells wheninfluenza A-infected Madin-Darby Canine Kidney (MDCK) cells were exposedto 425 nm light at various doses and a MOI for the WT-influenza A wasprovided at 0.1.

FIG. 98A is a chart showing the effectiveness of light at 405, 425, 450,and 470 nm and administered with a dose of 58.5 J/cm², in terms of hourspost-exposure, at killing P. aeruginosa.

FIG. 98B is a chart showing the effectiveness of light at 405, 425, 450,and 470 nm, and administered with a dose of 58.5 J/cm², in terms ofhours post-exposure, at killing S. aeurus.

FIG. 99A is a chart showing the effectiveness of light at 425 nm andadministered with doses ranging from 1 to 1000 J/cm² at killing P.aeruginosa.

FIG. 99B is a chart showing the effectiveness of light at 425 nm andadministered with doses ranging from 1 to 1000 J/cm² at killing S.aureus.

FIG. 100A is a chart showing the effectiveness of light at 405 nm andadministered with doses ranging from 1 to 1000 J/cm² at killing P.aeruginosa.

FIG. 100B is a chart showing the effectiveness of light at 405 nm andadministered with doses ranging from 1 to 1000 J/cm² at killing S.aureus.

FIG. 101 is a chart showing the toxicity of 405 nm and 425 nm light inprimary human aortic endothelial cells (HAEC).

FIG. 102A is a chart showing the bacterial logio reduction and the %loss of viability of infected AIR-100 tissues following exposure of thetissue to doses of light ranging from 4 to 512 J/cm² at 405 nm.

FIG. 102B is a chart showing the bacterial logio reduction and the %loss of viability of infected AIR-100 tissues following exposure of thetissue to doses of light ranging from 4 to 512 J/cm² at 425 nm.

FIG. 102C is a chart showing the bacterial logio reduction and the %loss of viability of infected AIR-100 tissues with gram negativebacteria (e.g., P. aeruginosa) following exposure of the tissue to dosesof light ranging from 4 to 512 J/cm² at 405 nm.

FIG. 102D is a chart showing the bacterial logio reduction and the %loss of viability of infected AIR-100 tissues with gram negativebacteria (e.g., P. aeruginosa) following exposure of the tissue to dosesof light ranging from 4 to 512 J/cm² at 425 nm.

FIG. 102E is a chart showing the bacterial logio reduction and the %loss of viability of infected AIR-100 tissues with gram positivebacteria (e.g., S. aureus) following exposure of the tissue to doses oflight ranging from 4 to 512 J/cm² at 405 nm.

FIG. 102F is a chart showing the bacterial logio reduction and the %loss of viability of infected AIR-100 tissues with gram positivebacteria (e.g., S. aureus) following exposure of the tissue to doses oflight ranging from 4 to 512 J/cm² at 425 nm.

FIGS. 103A-103J are a series of charts showing the effect of light at405 nm and 425 nm, at differing dosage levels, in terms of bacterialsurvival vs. dose (J/cm²) for both P. aeruginosa and S. aureus bacteria.

FIG. 104 is a table summarizing the light therapeutic index (LTI)calculations and corresponding bactericidal doses for the bacterialexperiments illustrated in FIGS. 79A-80.

FIG. 105 is a chart showing the effect of 425 nm light at various dosesat killing P. aeuriginosa over a period of time from 0 hours, 2 hours, 4hours, and 22.5 hours.

FIG. 106 is a chart showing that whether all of the light (J/cm²) isadministered in one dose or in a series of smaller doses, theantimicrobial effect (average CFU/ml) vs. dose (J/cm² X number oftreatments) is largely the same, at 8 hours and 48 hourspost-administration.

FIG. 107A is a chart showing the treatment of a variety ofdrug-resistant bacteria (Average CFU/ml) vs. dose (J/cm²) at 24 hourspost-exposure.

FIG. 107B is a table summarizing the tested bacteria species andstrains.

FIG. 107C is a table that summarizes the efficacy of twice daily dosingof 425 nm light against difficult-to-treat clinical lung pathogens.

DETAILED DESCRIPTION

The embodiments set forth below represent the necessary information toenable those skilled in the art to practice the embodiments andillustrate the best mode of practicing the embodiments. Upon reading thefollowing description in light of the accompanying drawing figures,those skilled in the art will understand the concepts of the disclosureand will recognize applications of these concepts not particularlyaddressed herein. It should be understood that these concepts andapplications fall within the scope of the disclosure and theaccompanying claims.

It should be understood that, although the terms first, second, etc. maybe used herein to describe various elements, these elements should notbe limited by these terms. These terms are only used to distinguish oneelement from another. For example, a first element could be termed asecond element, and, similarly, a second element could be termed a firstelement, without departing from the scope of the present disclosure. Asused herein, the term “and/or” includes any and all combinations of oneor more of the associated listed items.

It should be understood that when an element such as a layer, region, orsubstrate is referred to as being “on” or extending “onto” anotherelement, it can be directly on or extend directly onto the other elementor intervening elements may also be present. In contrast, when anelement is referred to as being “directly on” or extending “directlyonto” another element, there are no intervening elements present.Likewise, it will be understood that when an element such as a layer,region, or substrate is referred to as being “over” or extending “over”another element, it can be directly over or extend directly over theother element or intervening elements may also be present. In contrast,when an element is referred to as being “directly over” or extending“directly over” another element, there are no intervening elementspresent. It should also be understood that when an element is referredto as being “connected” or “coupled” to another element, it can bedirectly connected or coupled to the other element or interveningelements may be present. In contrast, when an element is referred to asbeing “directly connected” or “directly coupled” to another element,there are no intervening elements present.

It should be understood that, although the terms “upper,” “lower,”“bottom,” “intermediate,” “middle,” “top,” and the like may be usedherein to describe various elements, these elements should not belimited by these terms. These terms are only used to distinguish oneelement from another. For example, a first element could be termed an“upper” element and, similarly, a second element could be termed an“upper” element depending on the relative orientations of theseelements, without departing from the scope of the present disclosure.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the disclosure.As used herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises,”“comprising,” “includes,” and/or “including” when used herein specifythe presence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this disclosure belongs. It willbe further understood that terms used herein should be interpreted ashaving meanings that are consistent with their meanings in the contextof this specification and the relevant art and will not be interpretedin an idealized or overly formal sense unless expressly so definedherein.

Embodiments are described herein with reference to schematicillustrations of embodiments of the disclosure. As such, the actualdimensions of the layers and elements can be different, and variationsfrom the shapes of the illustrations as a result, for example, ofmanufacturing techniques and/or tolerances, are expected. For example, aregion illustrated or described as square or rectangular can haverounded or curved features, and regions shown as straight lines may havesome irregularity. Thus, the regions illustrated in the figures areschematic and their shapes are not intended to illustrate the preciseshape of a region of a device and are not intended to limit the scope ofthe disclosure. Additionally, sizes of structures or regions may beexaggerated relative to other structures or regions for illustrativepurposes and, thus, are provided to illustrate the general structures ofthe present subject matter and may or may not be drawn to scale. Commonelements between figures may be shown herein with common element numbersand may not be subsequently re-described.

Aspects of the present disclosure relate to devices and methods forimpinging light on a tissue, for example within a body of a patient,where the light may include at least one characteristic that exerts orinduces at least one biological effect within or on the tissue.Biological effects may include at least one of inactivating andinhibiting growth of one or more combinations of microorganisms andpathogens, including but not limited to viruses, bacteria, fungi, andother microbes, among others. Biological effects may also include one ormore of upregulating a local immune response, stimulating enzymaticgeneration of nitric oxide to increase endogenous stores of nitricoxide, releasing nitric oxide from endogenous stores of nitric oxide,and inducing an anti-inflammatory effect. Wavelengths of light may beselected based on at least one intended biological effect for one ormore of the targeted tissue and the targeted microorganisms orpathogens. In certain aspects, wavelengths of light may include visiblelight in any number of wavelength ranges based on the intendedbiological effect. Further aspects involve light impingement on tissuefor multiple microorganisms and/or multiple pathogenic biologicaleffects, either with light of a single peak wavelength or a combinationof light with more than one peak wavelength. Devices and methods forlight treatments are disclosed that provide light doses for inducingbiological effects on various targeted pathogens and targeted tissueswith increased efficacy and reduced cytotoxicity. Light doses mayinclude various combinations of irradiances, wavelengths, and exposuretimes, and such light doses may be administered continuously ordiscontinuously with a number of pulsed exposures.

Certain aspects of the disclosure relate to phototherapeutic modulationof nitric oxide in living mammalian tissue, including use of lighthaving a first peak wavelength and a first radiant flux to releasenitric oxide from endogenous stores of nitric oxide, and use of lighthaving a second peak wavelength and a second radiant flux to increaseendogenous stores of nitric oxide (e.g., to increase expression ofnitric oxide synthase enzymes), wherein the second peak wavelengthdiffers from the first peak wavelength. The photoinitiated release ofendogenous stores of nitric oxide effectively regenerates “gaseous” (orunbound) nitric oxide that was autooxidized into nitrosativeintermediates and bound covalently in the body in a “bound” state. Bystimulating release of nitric oxide from endogenous stores, nitric oxidemay be maintained in a gaseous state for an extended duration and/or aspatial zone of nitric oxide release may be expanded.

Certain aspects of the disclosure relate to phototherapeutic modulationof nitric oxide in living mammalian tissue, including use of lighthaving a first peak wavelength and a first radiant flux to stimulateenzymatic generation of nitric oxide to increase endogenous stores ofnitric oxide (e.g., to increase expression of nitric oxide synthaseenzymes), and release nitric oxide from the endogenous stores. Thephotoinitiated release of endogenous stores of nitric oxide effectivelyregenerates “gaseous” (or unbound) nitric oxide that was autooxidizedinto nitrosative intermediates and bound covalently in the body in a“bound” state. By stimulating release of nitric oxide from endogenousstores, nitric oxide may be maintained in a gaseous state for anextended duration and/or a spatial zone of nitric oxide release may beexpanded.

As noted previously, nitric oxide is endogenously stored on a variety ofnitrosated biochemical structures. Upon receiving the requiredexcitation energy, both nitroso and nitrosyl compounds undergo hemolyticcleavage of S-N, N-N, or M-N bonds to yield free radical nitric oxide.Nitrosothiols and nitrosamines are photoactive and can be phototriggeredto release nitric oxide by wavelength specific excitation. FIG. 2A is areaction sequence showing photomodulated release of NO fromnitrosothiols (RSNO). Similar results may be obtained with metalnitrosyls and NO-loaded chromophores (such as, but not limited to,CCO-NO). FIG. 2B is a reaction sequence showing photomodulated releaseof NO from metal nitrosyls (M-NO). FIG. 2C is a reaction sequenceshowing loading of cytochrome c oxidase (CCO) with NO (yielding CCO-NOand CCO-NO₂ ⁻), followed by photomodulated release of nitric oxide fromthe CCO-NO and CCO-NO₂ ⁻. In each case, providing light energy of aspecified peak wavelength and radiant flux to tissue may stimulaterelease of endogenous stores of NO to permit NO to be maintained in agaseous state in living tissue for a longer duration than would beencountered in the absence of the provision of such light energy.

FIG. 3 is a cross-sectional view of epidermis and dermis layers of humanskin with schematic illustration of overlapping zones 1-3 in whichendogenous stores of NO are generated and/or NO is released fromendogenous stores by photomodulation. (The zones 1-3 are not necessarilyillustrated to scale.) It has been reported that NO may diffuse inmammalian tissue by a distance of up to about 500 microns. In certainembodiments, photons of a first energy hυ₁ may be supplied to the tissueto stimulate enzymatic generation of NO to increase endogenous stores ofNO in a first diffusion zone 1. Photons of a second energy hue may besupplied to the tissue in a region within or overlapping the firstdiffusion zone 1 to trigger release of NO from endogenous stores,thereby creating a second diffusion zone 2. Alternatively, oradditionally, photons of a second energy hυ₂ may be supplied tostimulate enzymatic generation of NO to increase endogenous stores of NOin the second diffusion zone 2. Photons of a third energy hυ₃ may besupplied to the tissue in a region within or overlapping the seconddiffusion zone 2 to trigger release of endogenous stores, therebycreating a third diffusion zone 3. Alternatively, or additionally,photons of a third energy hυ₃ may be supplied to stimulate enzymaticgeneration of NO to increase endogenous stores of NO in the thirddiffusion zone 3. In certain embodiments, the first, second, and thirddiffusion zones 1-3 may have different average depths relative to anouter epidermal surface. In certain embodiments, the first photon energyhυ₁, the second photon energy hυ₂, and the third photon energy hυ₃ maybe supplied at different peak wavelengths, wherein different peakwavelengths may penetrate mammalian tissue to different depths—sincelonger wavelengths typically provide greater penetration depth. Incertain embodiments, sequential or simultaneous impingement ofincreasing wavelengths of light may serve to “push” a nitric oxidediffusion zone deeper within mammalian tissue than might otherwise beobtained by using a single (e.g., long) wavelength of light.

Light having a first peak wavelength and a first radiant flux to releasenitric oxide from endogenous stores of nitric oxide may be referred toherein as “endogenous store releasing light” or “ES releasing light;”and light having a second peak wavelength and a second radiant flux tostimulate enzymatic generation of nitric oxide to increase endogenousstores of nitric oxide may be referred to herein as “endogenous storeincreasing light” or “ES increasing light.”

In certain embodiments, the second peak wavelength (of the ES increasinglight) is greater than the first peak wavelength (of the ES releasinglight) by at least 25 nm, at least 40 nm, at least 50 nm, at least 60nm, at least 75 nm, at least 85 nm, at least 100 nm, or anotherthreshold specified herein.

In certain embodiments, each of the ES increasing light and the ESreleasing light has a radiant flux in a range of at least 5 mW, or atleast 10 mW, or at least 15 mW, or at least 20 mW, or at least 30 mW, orat least 40 mW, or at least 50 mW, or in a range of from 5 mW to 60 mW,or in a range of from 5 mW to 30 mW, or in a range of from 5 mW to 20mW, or in a range of from 5 mW to 10 mW, or in a range of from 10 mW to60 mW, or in a range of from 20 mW to 60 mW, or in a range of from 30 mWto 60 mW, or in a range of from 40 mW to 60 mW, or in another rangespecified herein.

In certain embodiments, the ES increasing light has a greater radiantflux than the ES releasing light. In certain embodiments, the ESreleasing light has a greater radiant flux than the ES increasing light.

In certain embodiments, one or both of the ES increasing light and theES releasing light has a radiant flux profile that is substantiallyconstant during a treatment window. In certain embodiments, at least oneof the ES increasing light and the ES releasing light has a radiant fluxprofile that increases with time during a treatment window. In certainembodiments, at least one of the ES increasing light and the ESreleasing light has a radiant flux profile that decreases with timeduring a treatment window. In certain embodiments, one of the ESincreasing light or the ES releasing light has a radiant flux profilethat decreases with time during a treatment window, while the other ofthe ES increasing light or the ES releasing light has a radiant fluxprofile that increases with time during a treatment window.

In certain embodiments, ES releasing light is applied to tissue during afirst time window, ES increasing light is applied to the tissue during asecond time window, and the second time window overlaps with the firsttime window. In other embodiments, ES releasing light is applied totissue during a first time window, ES increasing light is applied to thetissue during a second time window, and the second time isnon-overlapping or is only partially overlapping with the first timewindow. In certain embodiments, the second time window is initiated morethan one minute, more than 5 minutes, more than 10 minutes, more than 30minutes, or more than one hour after conclusion of the first timewindow. In certain embodiments, ES releasing light is applied to tissueduring a first time window, ES increasing light is applied to the tissueduring a second time window, and the first time window and the secondtime window are substantially the same. In other embodiments, the secondtime window is longer than the first time window.

In certain embodiments, one or both of ES increasing light and ESreleasing light may be provided by a steady state source providing aradiant flux that is substantially constant over a prolonged periodwithout being pulsed.

In certain embodiments, one or both of ES increasing light and ESreleasing light may include more than one discrete pulse (e.g., aplurality of pulses) of light. In certain embodiments, more than onediscrete pulse of ES releasing light is impinged on tissue during afirst time window, and/or more than one discrete pulse of ES increasinglight is impinged on tissue during a second time window. In certainembodiments, the first time window and the second time window may becoextensive, may be overlapping but not coextensive, or may benon-overlapping.

In certain embodiments, at least one of radiant flux and pulse durationof ES releasing light may be reduced from a maximum value to a non-zeroreduced value during a portion of a first time window. In certainembodiments, at least one of radiant flux and pulse duration of ESreleasing light may be increased from a non-zero value to a higher valueduring a portion of a first time window. In certain embodiments, atleast one of radiant flux and pulse duration of ES increasing light maybe reduced from a maximum value to a non-zero reduced value during aportion of a second time window. In certain embodiments, at least one ofradiant flux and pulse duration of ES increasing light may be increasedfrom a non-zero value to a higher value during a portion of a secondtime window.

In certain embodiments, each of ES increasing light and ES releasinglight consists of non-coherent light. In certain embodiments, each of ESincreasing light and ES releasing light consists of coherent light. Incertain embodiments, one of the ES increasing light or the ES releasinglight consists of non-coherent light, and the other of the ES increasinglight or the ES releasing light consists of coherent light.

In certain embodiments, the ES releasing light is provided by at leastone first light emitting device and the ES increasing light is providedby at least one second light emitting device. In certain embodiments,the ES releasing light is provided by a first array of light emittingdevices and the ES increasing light is provided by a second array oflight emitting devices.

In certain embodiments, at least one of the ES increasing light or theES releasing light is provided by at least one solid state lightemitting device. Examples of solid state light emitting devices include(but are not limited to) light emitting diodes, lasers, thin filmelectroluminescent devices, powdered electroluminescent devices, fieldinduced polymer electroluminescent devices, and polymer light-emittingelectrochemical cells. In certain embodiments, the ES releasing light isprovided by at least one first solid state light emitting device and theES increasing light is provided by at least one second solid state lightemitting device. In certain embodiments, ES increasing light and ESreleasing light may be generated by different emitters contained in asingle solid state emitter package, wherein close spacing betweenadjacent emitters may provide integral color mixing. In certainembodiments, the ES releasing light may be provided by a first array ofsolid state light emitting devices and the ES increasing light may beprovided by a second array of solid state light emitting devices. Incertain embodiments, an array of solid state emitter packages eachincluding at least one first emitter and at least one second emitter maybe provided, wherein the array of solid state emitter packages embodiesa first array of solid state emitters arranged to generate ES releasinglight and embodies a second array of solid state emitters arranged togenerate ES increasing light. In certain embodiments, an array of solidstate emitter packages may embody packages further including third,fourth, and/or fifth solid state emitters, such that a single array ofsolid state emitter packages may embody three, four, or five arrays ofsolid state emitters, wherein each array is arranged to generateemissions with a different peak wavelength.

In certain embodiments, at least one of the ES increasing light or theES releasing light is provided by at least one light emitting devicedevoid of a wavelength conversion material. In other embodiments, atleast one of the ES increasing light or the ES releasing light isprovided by at least one light emitting device arranged to stimulate awavelength conversion material, such as a phosphor material, afluorescent dye material, a quantum dot material, and a fluorophorematerial.

In certain embodiments, the ES increasing light and the ES releasinglight consist of substantially monochromatic light. In certainembodiments, the ES releasing light includes a first spectral outputhaving a first full width at half maximum value of less than 25 nm (orless than 20 nm, or less than 15 nm, or in a range of from 5 nm to 25nm, or in a range of from 10 nm to 25 nm, or in a range of from 15 nm to25 nm), and/or the ES increasing light includes a second spectral outputhaving a second full width at half maximum value of less than 25 nm (orless than 20 nm, or less than 15 nm, or in a range of from 5 nm to 25nm, or in a range of from 10 nm to 25 nm, or in a range of from 15 nm to25 nm). In certain embodiments, less than 5% of the first spectraloutput is in a wavelength range of less than 400 nm, and less than 1% ofthe second spectral output is in a wavelength range of less than 400 nm.

In certain embodiments, the ES releasing light is produced by one ormore first light emitters having a single first peak wavelength, and theES increasing light is produced by one or more second light emittershaving a single second peak wavelength. In other embodiments, the ESincreasing light may be produced by at least two light emitters havingdifferent peak wavelengths (e.g., differing by at least 5 nm, at least10 nm, at least 15 nm, at least 20 nm, or at least 25 nm), and/or the ESreleasing light may be produced by at least two light emitters havingdifferent peak wavelengths (e.g., differing by at least 5 nm, at least10 nm, at least 15 nm, at least 20 nm, or at least 25 nm).

Ultraviolet light (e.g., UV-A light having a peak wavelength in a rangeof from 350 nm to 395 nm, and UV-B light having a peak wavelength in arange of from 320 nm to 350 nm) may be effective as ES increasing or ESreleasing light; however, overexposure to ultraviolet light may lead todetrimental health effects including premature skin aging andpotentially elevated risk for certain types of cancer. In certainembodiments, UV light (e.g., having peak wavelengths in a range of from320 nm to 399 nm) may be used as ES increasing or ES releasing light;however, in other embodiments, UV light may be avoided.

In certain embodiments, ES increasing light and ES releasing light aresubstantially free of UV light. In certain embodiments, less than 5% ofthe ES increasing light is in a wavelength range of less than 400 nm,and less than 1% of the ES releasing light output is in a wavelengthrange of less than 400 nm. In certain embodiments, ES increasing lightincludes a peak wavelength in a range of from 400 nm to 490 nm, or from400 nm to 450 nm, or from 400 nm to 435 nm, or from 400 nm to 420 nm, orfrom 410 nm to 440 nm, or from 420 nm to 440 nm.

In certain embodiments, ES increasing light may include a wavelengthrange and flux that may alter the presence, concentration, or growth ofpathogens (e.g., bacteria, viruses, fungi, protists, and/or othermicrobes) in or on living mammalian tissue receiving the light. UV lightand near-UV light (e.g., having peak wavelengths from 400 nm to 435 nm,or more preferably from 400 nm to 420 nm) in particular may affectmicrobial growth. Effects on microbial growth may depend on thewavelength range and dose. In certain embodiments, ES increasing or ESreleasing light may include near-UV light having a peak wavelength in arange of from 400 nm to 420 nm to provide a bacteriostatic effect (e.g.,with pulsed light having a radiant flux of <9 mW), provide abactericidal effect (e.g., with substantially steady state light havinga radiant flux in a range of from 9 mW to 17 mW), or provide anantimicrobial effect (e.g., with substantially steady state light havinga radiant flux in a range of greater than 17 mW, such as in a range offrom 18 mW to 60 mW). In certain embodiments, ES increasing or ESreleasing light in a near-UV range (e.g., from 400 nm to 420 nm) mayalso affect microbial growth (whether in a bacteriostatic range,bactericidal range, or an antimicrobial range) for uses such as woundhealing, reduction of acne blemishes, or treatment of atopic dermatitis.Such function(s) may be in addition to the function of the ES increasinglight to increase endogenous stores of nitric oxide in living tissue.

In certain embodiments, ES increasing light may include a peakwavelength in a range of from 500 nm to 900 nm, or in a range of from490 nm to 570 nm, or in a range of from 510 nm to 550 nm, or in a rangeof from 520 nm to 540 nm, or in a range of from 525 nm to 535 nm, or ina range of from 528 nm to 532 nm, or in a range of about 530 nm.

Applicant has found that the ability to generate and release nitricoxide is dependent on the wavelength and fluence of light used. Toinvestigate whether certain wavelengths of light may be more effectivethan others at releasing endogenous stores of NO (i.e., to serve as ESreleasing light), Applicant performed various experiments. One series ofexperiments included generating nitric oxide-loaded hemoglobin (Hb-NO),irradiating the Hb-NO with different wavelengths of light produced bysubstantially monochromatic LEDs, and comparing absorbance spectra forHb, for the Hb-NO prior to the LED irradiation, and for the Hb-NO afterthe LED irradiation. The Hb-NO was generated by mixing 10 μM Hb with 1μM Prolino (a NO source). The mixture was then stirred and incubated onehour, and then was allowed to rest for 5 minutes. Irradiation with LEDlight was performed under vacuum to facilitate removal of NO liberatedfrom the Hb-NO.

FIG. 4A includes superimposed plots of absorbance versus wavelength forhemoglobin (Hb) (line “A1”), for nitric oxide-loaded hemoglobin (Hb-NO)prior to irradiation (line “B1”), and for Hb-NO following absorption of150 J of light emissions of a green LED having a peak wavelength of 530nm (line “Cl”). A comparison of line Al and line B1 shows the presenceof two peaks at about 540 nm and about 577 nm, representing the presenceof NO in the Hb-NO. A comparison of line Cl and line B1 shows that thetwo peaks at about 540 nm and about 577 nm present in the Hb-NO wereeliminated, thereby evidencing release of NO from the Hb-NO attributableto irradiation of Hb-NO with 530 nm peak wavelength green light.

FIG. 4B includes superimposed plots of absorbance versus wavelength forHb (line “A2”), for Hb-NO prior to irradiation (line “B2”), and forHb-NO following absorption of 150 J of light emissions of an IR LEDsource having a peak wavelength of 850 nm (line “C2”). A comparison ofline A2 and line B2 shows the presence of two peaks at about 540 nm andabout 577 nm, representing the presence of NO in the Hb-NO. A comparisonof lines C1 and B1, however, reveals that such lines substantiallycoincide with one another. This evidences that impingement of 850 nmpeak wavelength light on Hb-NO was ineffective in releasing NO.

Nine LED light sources providing nine different peak wavelengths (i.e.,410 nm, 447 nm, 470 nm, 505 nm, 530 nm, 597 nm, 630 nm, 660 nm, and 850nm) were tested to determine their relative effectiveness in releasingNO from Hb-NO. FIG. 5 is a plot of percentage change in peak absorbancefor the 540 nm peak of Hb-NO versus fluence (Joules per squarecentimeter) for the nine different wavelengths of light from 410 nm to850 nm. As shown in FIG. 5, the wavelengths identified to be mosteffective in releasing NO from Hb-NO were determined to be thefollowing, from best to worst: 530 nm, 505 nm, 597 nm, 447 nm, 660 nm,470 nm, 410 nm, 630 nm, and 850 nm.

Another series of experiments included generating nitric oxide-loadedcytochrome c (Cytochrome c-NO), irradiating the Cytochrome c-NO withdifferent wavelengths of light produced by substantially monochromaticLEDs, and comparing absorbance spectra for Cytochrome c, for theCytochrome c-NO prior to the LED irradiation, and for the Cytochromec-NO after the LED irradiation. 60 μM Cytochrome c was used according toa procedure otherwise similar to that described above in connection withHb. FIG. 6 is a plot of percentage change in peak absorbance forCytochrome c-NO versus fluence (Joules per square centimeter) for ninedifferent wavelengths of light (from 410 nm to 850 nm). As shown in FIG.6, the wavelengths identified to be most effective in releasing NO fromCytochrome c-NO were determined to be the following, from best to worst:530 nm, 597 nm, 505 nm, 660 nm, 470 nm, 630 nm, 410 nm, 447 nm, and 850nm. Notably, 530 nm was determined to be the most effective peakwavelength of light for releasing NO from both Hb-NO and Cytochromec-NO.

The results shown in FIG. 5 for Hb-NO were not normalized to radiantflux, and it is recognized that different LEDs have differentefficiencies. To address this issue, the results for Hb-NO werenormalized to a 300 mA value. FIG. 7 is a plot of released NO permilliwatt per square centimeter versus time for the photomodulatedrelease of NO from Hb-NO for nine different wavelengths of light (from410 nm to 850 nm). As shown in FIG. 7, 530 nm was determined to be thesingle most efficient single peak wavelength (per milliwatt of power)for releasing NO from Hb-NO.

To determine whether various combinations of two peak wavelengths mightbe more or less effective than single peak wavelengths in releasing NOfrom Hb-NO, additional experiments were performed using Hb-NO. Hb-NO wasgenerated by mixing 10 μM Hb with 1 μM Prolino (a NO source), then themixture was stirred and incubated one hour, and the mixture was allowedto rest for 5 minutes. Irradiation with two peak wavelengths of LEDlight was performed under vacuum to facilitate removal of NO liberatedfrom the Hb-NO. Results for three different combinations of two peakwavelengths are shown in FIGS. 8A to 8C, together with results obtainedfor individual constituents of the combinations.

FIG. 8A includes superimposed plots of released NO per milliwatt persquare centimeter versus time for irradiation of Hb-NO with (i) a 410 nmpeak wavelength blue LED device, (ii) a 530 nm peak wavelength green LEDdevice, and (iii) the 410 nm peak wavelength blue LED device incombination with the 530 nm peak wavelength green LED device. Asexpected from the prior experiments, the 410 nm light was significantlyless effective than the 530 nm light at releasing NO from Hb-NO.Surprisingly, however, the combination of equal parts of 410 nm lightand 530 nm light appeared to be equally as effective as 530 nm lightalone. Such a combination may be beneficial since a 410 nm blue LED issignificantly more efficient than a 530 nm green LED, such that acombination of equal parts of 410 nm LED emissions and 530 nm LEDemissions may use 26% less electric power than emissions of a 530 nm LEDalone, when operated to provide the same radiant flux.

FIG. 8B includes superimposed plots of released NO per milliwatt persquare centimeter versus time for irradiation of Hb-NO with (i) a 530 nmpeak wavelength green LED device, (ii) a 660 nm peak wavelength red LEDdevice, and (iii) the 530 nm peak wavelength green LED device incombination with the 660 nm peak wavelength red LED device. As expectedfrom the prior experiments, the 660 nm red light was significantly lesseffective than the 530 nm green light at releasing NO from Hb-NO. Thecombination of equal parts of 530 nm green light and 660 nm red lightwas only slightly better than 660 nm red light alone at releasing NOfrom Hb-NO.

Notably, as shown in FIG. 8B, the release of NO from Hb-NO appears to bethe same for 530 nm green light, 660 nm red light, and a combination of530 nm green and 660 nm light for the time window of from 0 seconds toabout 2000 seconds, but the effectiveness of the different sourcesdiverges thereafter. Without intending to be bound by any particulartheory or explanation of this phenomenon, it is suggested that NO bindsto Hb-NO at multiple sites, and that removal of a second or subsequentNO molecule from Hb-NO may require more energy than removal of a firstNO molecule, perhaps due to a change in shape of the Hb-NO after removalof a first NO molecule.

FIG. 8C includes superimposed plots of released NO per milliwatt persquare centimeter versus time for irradiation of Hb-NO with (i) a 530 nmpeak wavelength green LED device (including one repeat run), (ii) a 850nm peak wavelength infrared LED device (including one repeat run), and(iii) the 530 nm peak wavelength green LED device in combination withthe 850 nm peak wavelength infrared LED device. As expected from theprior experiments, the 850 nm infrared light was ineffective atreleasing NO from Hb-NO. The combination of equal parts of 530 nm greenlight and 850 nm infrared light was also ineffective at releasing NOfrom Hb-NO. This shows that the addition of 530 nm green light wasunable to enhance the effectiveness of 850 nm infrared light inreleasing NO from Hb-NO.

In certain embodiments, ES releasing light that includes light having afirst peak wavelength is impinged on living tissue, ES increasing lightthat includes light having a second peak wavelength is impinged on theliving tissue, and furthermore a light having a third peak wavelengthmay be impinged on the living tissue. In certain embodiments, the lighthaving a third peak wavelength may be provided at substantially the sametime as (or during a time window overlapping at least one time windowof) one or both of the ES increasing light and the ES releasing light.In certain embodiments, the light having a third peak wavelength differsfrom each of the first peak wavelength and the second peak wavelength byat least 10 nm. In certain embodiments, the light having a third peakwavelength exceeds the second peak wavelength by at least 20 nm. Incertain embodiments, the light having a third peak wavelength isprovided with a radiant flux in a range of from 5 mW to 60 mW. Incertain embodiments, the third peak wavelength is in a range of from 600nm to 900 nm, or in a range of from 600 nm to 700 nm. In certainembodiments, the third peak wavelength is in a range of from 320 nm to399 nm.

In certain embodiments, light having a third peak wavelength in a rangeof from 620 nm to 670 nm (e.g., including specific wavelengths of about630 nm and about 660 nm) may be useful to provide anti-inflammatoryeffects and/or to promote vasodilation. Anti-inflammatory effects may beuseful to promote wound healing, to reduce acne blemishes, to promotefacial aesthetics, and/or to treat atopic dermatitis and other topicaldermatological disorders. Vasodilation may also be beneficial to treatandrogenic alopecia or other topical dermatological disorders.

In certain embodiments, light having a third peak wavelength may beuseful to promote thermal and/or infrared heating of living mammaliantissue, such as may be useful in certain contexts including woundhealing.

In certain embodiments utilizing modulated light therapy to control NOgeneration and release, human immune response may be altered and/orcontrolled. Such responses may include, but are not limited to: ATPproduction; DNA and RNA synthesis; gene transcription; extracellularmatrix (e.g., collagen and elastin) secretion; protein expression(including but not limited to NOS enzymes); cell signaling pathways(including cytokine expression (e.g., interleukins), growth factors(e.g., vascular endothelial growth factor, insulin growth factor,insulin-like growth factors, fibroblast growth factors, and tumornecrosis factors); Wnt signaling pathways; and NF-kB pathways); cellularviability; cellular apoptosis; cellular proliferation and migration;reactive oxygen species generation; cellular response to reactive oxygenspecies (e.g., expression of superoxide dismutase); and inhibition ofthe enzyme 5α-reductase (to decrease DHT production and thereby reduceor reverse hair loss).

Methods and devices disclosed herein for photomodulation of nitric oxidein living mammalian tissue are contemplated for use with a wide varietyof tissues. In certain embodiments, the tissue comprises epithelialtissue. In certain embodiments, the tissue comprises mucosal tissue. Incertain embodiments, the tissue is within a body cavity of a patient. Incertain embodiments, the tissue comprises cervical tissue.

In certain embodiments, the impinging of light having a first peakwavelength and the impinging of light having a second peak wavelength isperformed with a single therapeutic device.

In certain embodiments, a device for photomodulation of nitric oxide inliving mammalian tissue as disclosed herein may include a flexiblesubstrate supporting one or more light emitting elements and arranged toconform to at least a portion of a human body. In certain embodiments, aflexible substrate may include a flexible printed circuit board (PCB),such as may include at least one polyimide-containing layer and at leastone layer of copper or another electrically conductive material. Inother embodiments, a device for photomodulation of nitric oxide asdisclosed herein may include a rigid substrate supporting one or morelight emitting elements. In certain embodiments, one or more surfaces ofa device for photomodulation of nitric oxide may include alight-transmissive encapsulant material arranged to cover any lightemitter(s) and at least a portion of an associated substrate (e.g.,flexible PCB). A preferred encapsulant material is silicone, which maybe applied by any suitable means such as molding, dipping, spraying,dispensing, or the like. In certain embodiments, one or more functionalmaterials may be added to or coated on an encapsulant material. Incertain embodiments, at least one surface, or substantially all surfaces(e.g., front and back surfaces) of a flexible PCB may be covered withencapsulant material.

In certain embodiments, a substrate as described herein may be arrangedto support one or more light emitting elements. In certain embodiments,one or more light emitting elements may include multi-emitting lightemitting devices such as multi-LED packages. In certain embodiments, oneor more light emitting elements may be arranged for direct illumination,wherein at least a portion of emissions generated by the one or morelight emitting elements is arranged to be transmitted directly through alight-transmissive external surface of a device without need for anintervening waveguide or reflector. In certain embodiments, one or morelight emitting elements may be arranged for indirect illumination (e.g.,side illumination), wherein emissions generated by the one or more lightemitting elements are arranged to be transmitted to a light-transmissiveexternal surface via a waveguide and/or a reflector, without a lightemitting element being in direct line-of-sight arrangement relative to alight-transmissive external surface. In certain embodiments, a hybridconfiguration may be employed, including one or more light emittingelements arranged for direct illumination, and further including one ormore light emitting elements arranged for indirect illumination. Incertain embodiments, one or more reflective materials (e.g., reflectiveflexible PCB or other reflective films) may be provided along selectedsurfaces of a device to reduce internal absorption of light and todirect light emissions toward an intended light-transmissive surface. Incertain embodiments, a flexible light emitting device may include asubstantially uniform thickness. In other embodiments, a flexible lightemitting device may include a thickness that varies with position, suchas a thickness that tapers in one direction or multiple directions. Incertain embodiments, presence of a tapered thickness may help a flexiblelight emitting device to more easily be wrapped against or to conform toareas of a mammalian (e.g., human) body.

In certain embodiments, one or multiple holes or perforations may bedefined in a substrate and any associated encapsulant material. Incertain embodiments, holes may be arranged to permit transit of air,such as may be useful for thermal management. In certain embodiments,holes may be arranged to permit transit of wound exudate. In certainembodiments, one or more holes may be arranged to permit sensing of atleast one condition through the hole(s). Holes may be defined by anysuitable means such as laser perforation, die pressing, slitting,punching, blade cutting, and roller perforation. In certain embodiments,holes may have uniform or non-uniform size, placement, and/ordistribution relative to a substrate and encapsulant material.

In certain embodiments, a device for photomodulation of nitric oxide inliving mammalian tissue as disclosed herein may include one or morelight-affecting elements such as one or more light extraction features,wavelength conversion materials, light diffusion or scatteringmaterials, and/or light diffusion or scattering features. In certainembodiments, one or more light affecting elements may be arranged in alayer between a light emitting element and a light transmissive surfaceof a device. In certain embodiments, an encapsulant material (e.g.,encapsulant material layer) may be arranged between at least one lightemitting element and one or more light affecting elements. In certainembodiments, one or more light affecting elements may be formed ordispersed within an encapsulant material.

In certain embodiments, impingement of light on living tissue and/oroperation of a device as disclosed herein may be responsive to one ormore signals generated by one or more sensors or other elements. Varioustypes of sensors are contemplated, including temperature sensors,photosensors, image sensors, proximity sensors, pressure sensors,chemical sensors, biosensors, accelerometers, moisture sensors,oximeters, current sensors, voltage sensors, and the like. Otherelements that may affect impingement of light and/or operation of adevice as disclosed herein include a timer, a cycle counter, a manuallyoperated control element, a wireless transmitter and/or receiver (as maybe embodied in a transceiver), a laptop or tablet computer, a mobilephone, or another portable electronic or digital device external to alighting device. Wired and/or wireless communication between a device asdisclosed herein and one or more signal generating or signal receivingelements may be provided.

In certain embodiments, impingement of light on living tissue and/oroperation of a device as disclosed herein may be responsive to one ormore temperature signals. For example, a temperature condition may besensed on or proximate to (a) a device arranged to emit ES increasinglight and/or ES releasing light or (b) the tissue; at least one signalindicative of the temperature condition may be generated; and operationof a lighting device may be controlled responsive to the at least onesignal. Such control may include initiation of operation, deviation (oralteration) of operation, or termination of operation of light emittingelements, such as elements arranged to emit ES increasing light and/orES releasing light. In certain embodiments, thermal foldback protectionmay be provided at a threshold temperature (e.g., >42° Celsius) toprevent a user from experiencing burns or discomfort. In certainembodiments, thermal foldback protection may trigger a light emittingdevice to terminate operation, reduce current, or change an operatingstate in response to receipt of a signal indicating an excesstemperature condition.

In certain embodiments, a device for modulating nitric oxide in livingmammalian tissue as disclosed herein may be used for wound care, and mayinclude one or more sensors. In certain embodiments, one or more lightemitters and photodiodes may be provided to illuminate a wound site withone or more selected wavelengths (e.g., green light) to detect bloodflow in or proximate to the wound site to provide photoplethsmyographydata. One sensor or multiple sensors may be provided. A device mayalternatively or additionally include sensors arranged to detect bloodpressure, bandage or dressing covering pressure, heart rate,temperature, presence or concentration of chemical or biological species(e.g., in wound exudate), or other conditions.

In certain embodiments, a device for modulating nitric oxide in livingmammalian tissue as disclosed herein may include a memory element tostore information indicative of one or more sensor signals. Suchinformation may be used for diagnosis, assessing patient compliance,assessing patient status, assessing patient improvement, and assessingfunction of the device. In certain embodiments, information indicativeof one or more sensor signals may be transmitted via wired or wirelessmeans (e.g., via Bluetooth, WiFi, Zigbee, or another suitable protocol)to a mobile phone, a computer, a data logging device, or anothersuitable device that may optionally be connected to a local network, awide-area network, a telephonic network, or other communication network.In certain embodiments, a data port (e.g., micro USB or other type) maybe provided to permit extraction or interrogation of informationcontained in a memory.

Details of illustrative devices that may be used for modulating nitricoxide in living mammalian tissue are described hereinafter.

FIG. 9 is a side cross-sectional schematic view of a portion of a device10 for delivering light energy to living mammalian tissue, the device 10including multiple direct view light emitting sources 12 supported by asubstrate 11 and covered with an encapsulant material 14, which may beembodied in a sheet or layer. The substrate 11 preferably includes aflexible PCB, which may include a reflective surface to reflect lighttoward a light-transmissive outer surface 19 of the device 10. As shownin FIG. 9, the encapsulant material 14 covers the light emitting sources12 and an upper surface of the substrate 11; however, it is to beappreciated that in certain embodiments the encapsulant material 14 maycover both upper and lower surfaces of the substrate 11. In certainembodiments, different light emitting sources 12 may generate lighthaving different peak wavelengths. In certain embodiments, one or morelight emitting sources 12 may include a multi-emitter package arrangedto generate one or multiple peak wavelengths of light. In certainembodiments, one or more light emitting sources 12 may be arranged toproduce one or both of ES increasing light and ES releasing light.

FIG. 10 is a side cross-sectional schematic view of a portion of adevice 20 for delivering light energy to living mammalian tissue, thedevice 20 including multiple direct view light emitting sources 22supported by a substrate 21 and covered with an encapsulant material 24,which may be embodied in a sheet or layer. The substrate 21 preferablyincludes a flexible PCB, which may include a reflective surface toreflect light toward a light-transmissive outer surface 29 of the device20. At least one functional material (e.g., wavelength conversionmaterial and/or scattering material) 23 is disposed within theencapsulant material 24. In certain embodiments, the at least onefunctional material 23 includes one or more wavelength conversionmaterials, such as at least one of a phosphor material, a fluorescentdye material, a quantum dot material, and a fluorophore material. Incertain embodiments, wavelength materials of different peak wavelengthsmay be applied over different light emitting sources 22. In certainembodiments, the at least one functional material 23 is applied bydispensing or printing. In certain embodiments, one or more lightemitting sources 22 may include a multi-emitter package arranged togenerate one or multiple peak wavelengths of light. In certainembodiments, one or more light emitting sources 22 may be arranged toproduce one or both of ES increasing light and ES releasing light.

FIG. 11 is a side cross-sectional schematic view of a portion of adevice 30 for delivering light energy to living mammalian tissue, thedevice 30 including multiple direct view light emitting sources 32supported by a substrate 31 and covered with two encapsulant materiallayers 34A, 34B, with at least one functional material (e.g., wavelengthconversion and/or scattering material) sheet or layer 33 disposedbetween the encapsulant material layers 34A, 34B. The substrate 31preferably includes a flexible PCB, which may include a reflectivesurface to reflect light toward a light-transmissive outer surface 39 ofthe device 30. In certain embodiments, the at least one functionalmaterial sheet or layer 33 includes one or more wavelength conversionmaterials, such as at least one of a phosphor material, a fluorescentdye material, a quantum dot material, or a fluorophore material. Incertain embodiments, one or more light emitting sources 32 may include amulti-emitter package arranged to generate one or multiple peakwavelengths of light. In certain embodiments, one or more light emittingsources 32 may be arranged to produce one or both of ES increasing lightand ES releasing light.

FIG. 12 is a side cross-sectional schematic view of a portion of adevice 40 for delivering light energy to living mammalian tissue, thedevice 40 including multiple direct view light emitting sources 42supported by a substrate 41 and covered by an encapsulant material 44,which may be embodied in a sheet or layer. The substrate 41 preferablyincludes a flexible PCB, which may include a reflective surface toreflect light toward a light-transmissive outer surface 49 of the device40. The encapsulant material 44 is covered with a diffusion orscattering material layer 43. In certain embodiments, the diffusion orscattering material layer 43 may include acrylic, PET-G, silicone, or apolymeric sheet. In certain embodiments, the diffusion or scatteringmaterial layer 43 may include scattering particles such as zinc oxide,silicon dioxide, titanium dioxide, or the like. In certain embodiments,one or more light emitting sources 42 may include a multi-emitterpackage arranged to generate one or multiple peak wavelengths of light.In certain embodiments, one or more light emitting sources 42 may bearranged to produce one or both of ES increasing light and ES releasinglight.

FIG. 13 is a side cross-sectional schematic view of a portion of adevice 50 for delivering light energy to living mammalian tissue, thedevice 50 including multiple direct view light emitting sources 52supported by a substrate 51. The substrate 51 preferably includes aflexible PCB, which may include a reflective surface to reflect lighttoward a light-transmissive outer surface 59 of the device 50. Multiplemolded features 55 (e.g., molded from silicone) overlie the lightemitting sources 52. An encapsulant or light coupling material 54 isarranged between the light emitting sources 52 and the molded features55. In certain embodiments, light coupling material 54 may include alight coupling gel with an index of refraction that differs from anindex of refraction of the molded features 55. The molded features 55may be arranged along the light transmissive outer surface 59 of thedevice 50. In certain embodiments, one or more light emitting sources 52may include a multi-emitter package arranged to generate one or multiplepeak wavelengths of light. In certain embodiments, one or more lightemitting sources 52 may be arranged to produce one or both of ESincreasing light and ES releasing light.

FIG. 14 is a side cross-sectional schematic view of a portion of adevice 60 for delivering light energy to living mammalian tissue, thedevice 60 including a flexible substrate 61, a passive-matrix organiclight emitting diode (OLED) structure (embodied in an anode layer 66A, acathode layer 66B, and an OLED stack 62 between the anode and cathodelayers 66A, 66B. In certain embodiments, the OLED stack 62 may beconfigured to generate multiple wavelengths of light. The substrate 61preferably includes a flexible PCB, which may include a reflectivesurface to reflect light toward a light-transmissive outer surface 69 ofthe device 60. An encapsulant layer 64 is arranged over the cathodelayer 66B and preferably defines the light-transmissive outer surface 69of the device 60. In certain embodiments, one or more light emittingwavelengths produced by the OLED stack 62 may include ES increasinglight and/or ES releasing light.

FIG. 15 is a side cross-sectional schematic view of a portion of adevice 70 for delivering light energy to living mammalian tissue, thedevice 70 including a flexible substrate 71, multiple direct view lightemitting sources 72 supported by the substrate 71, and encapsulantmaterial layers 74A, 74B arranged above and below the substrate 71,respectively. The substrate 71 preferably includes a flexible PCB, whichmay include a reflective surface to reflect light toward alight-transmissive outer surface 79 of the device 70. The light emittingdevice 70 further includes holes or perforations 77 defined through boththe substrate 71 and the encapsulant material layers 74A, 74B. Incertain embodiments, one or more light emitting sources 72 may bearranged to produce one or both of ES increasing light and ES releasinglight.

FIG. 16 is a side cross-sectional schematic view of a portion of adevice 80 for delivering light energy to living mammalian tissue,wherein the device 80 includes multiple direct view light emittingsources 82 supported by a flexible substrate 81 and covered by anencapsulant layer 84. The substrate 81 preferably includes a flexiblePCB, which may include a reflective surface to reflect light toward alight-transmissive outer surface 89 of the device 80. The device 80 ispreferably flexible to permit it to be bent or shaped into a variety ofshapes to conform to a portion of a mammalian body. As illustrated, thedevice 80 is arranged in a concave configuration with the multiple lightemitting sources 82 arranged to direct emissions toward a center ofcurvature of the device 80. In certain embodiments, one or more lightemitting sources 82 may be arranged to produce one or both of ESincreasing light and ES releasing light.

FIG. 17 is a side cross-sectional schematic view of a portion of adevice 90 for delivering light energy to living mammalian tissue,wherein the device 90 includes multiple direct view light emittingsources 92 supported by a flexible substrate 91 and covered by anencapsulant layer 94. The substrate 91 preferably includes a flexiblePCB, which may include a reflective surface to reflect light toward alight-transmissive outer surface 99 of the device 90. The device 90 ispreferably flexible to permit it to be bent or shaped into a variety ofshapes to conform to a portion of a mammalian body. As illustrated, thedevice 90 is arranged in a convex configuration with the multiple lightemitting elements 92 arranged to direct emissions away from a center ofcurvature of the device 90. In certain embodiments, one or more lightemitting sources 92 may be arranged to produce one or both of ESincreasing light and ES releasing light.

FIG. 18 is a side cross-sectional schematic view of a portion of adevice 100 for delivering light energy to living mammalian tissue,wherein the device 100 is edge lit with one or more light emittingsources 102 supported by a flexible printed circuit board (PCB) 101 thatpreferably includes a reflective surface. Other non-light-transmissivesurfaces of the device 100 are bounded by a flexible reflectivesubstrate 105 arranged to reflect light toward a light-transmissiveouter surface 109 of the device 100. The flexible PCB 101, the lightemitting source(s) 102, and the flexible reflective substrate 105 arecovered with an encapsulant material 104, which may include silicone. Asillustrated, the device 100 may include a substantially constantthickness. In certain embodiments, one or more light emitting sources102 may be arranged to produce one or both of ES increasing light and ESreleasing light.

FIG. 19 is a side cross-sectional schematic view of a portion of adevice 110 for delivering light energy to living mammalian tissue,wherein the device 110 is edge lit with one or more light emittingsources 112 supported by a flexible PCB 111 that preferably includes areflective surface. A non-light-transmitting face of the device 110 isbounded by a flexible reflective substrate 115 arranged to reflect lighttoward a light-transmissive outer surface 119 of the device 110. Theflexible PCB 111, the light emitting source(s) 112, and the flexiblereflective substrate 115 are covered with an encapsulant material 114,which may include silicone. As illustrated, the device 110 may include athickness that is tapered with distance away from the light emittingsources 112. Such tapered thickness may enable the device 110 to moreeasily be wrapped against or to conform to areas of a mammalian (e.g.,human) body. In certain embodiments, one or more light emitting sources112 may be arranged to produce one or both of ES increasing light and ESreleasing light.

FIG. 20 is a side cross-sectional schematic view of a portion of adevice 120 for delivering light energy to living mammalian tissue,wherein the device 120 is edge lit with one or more light emittingsources 122 supported by a flexible PCB 121 that bounds multiple edgesand a face of the device 120. The flexible PCB 121 preferably includes areflective surface arranged to reflect light toward a light-transmissiveouter surface 129 of the device 120. The flexible PCB 121 and the lightemitting source(s) 122 are covered with an encapsulant material 124,which may include silicone. In certain embodiments, one or more lightemitting sources 122 may be arranged to produce one or both of ESincreasing light and ES releasing light.

FIG. 21 is a side cross-sectional schematic view of a portion of adevice 130 for delivering light energy to living mammalian tissue,wherein the device 130 is edge lit with one or more light emittingsources 132 supported by a flexible PCB 131 that bounds one edge and oneface of the device 130. The flexible PCB 131 preferably includes areflective surface arranged to reflect light toward a light-transmissiveouter surface 139 of the device 130. The flexible PCB 131 and the lightemitting source(s) 132 are covered with an encapsulant material 134,which may include silicone. As illustrated, the device 130 may include athickness that is tapered with distance away from the light emittingsources 132. In certain embodiments, one or more light emitting sources132 may be arranged to produce one or both of ES increasing light and ESreleasing light.

FIG. 22 is a side cross-sectional schematic view of a portion of adevice 140 for delivering light energy to living mammalian tissue,wherein the device 140 is edge lit with one or more light emittingsources 142 supported by a flexible PCB 141 that bounds multiple edgesand a face of the device 140. In certain embodiments, one or more lightemitting sources 142 may include a multi-emitter package arranged togenerate one or multiple peak wavelengths of light. The flexible PCB 141preferably includes a reflective surface arranged to reflect lighttoward a light-transmissive outer surface 149 of the device 140. Theflexible PCB 141 and the light emitting source(s) 142 are covered withan encapsulant material 144, which may include silicone. Between thelight-transmissive outer surface 149 and the encapsulant material 144,the device 140 further includes a diffusing and/or scattering layer 143.In certain embodiments, the diffusing and/or scattering layer 143 mayinclude a sheet of material; in other embodiments, the diffusing and/orscattering layer 143 may include particles applied in or on theencapsulant material 144. In certain embodiments, one or more lightemitting sources 142 may be arranged to produce one or both of ESincreasing light and ES releasing light.

FIG. 23 is a side cross-sectional schematic view of a portion of adevice 150 for delivering light energy to living mammalian tissue,wherein the device 150 is edge lit with one or more light emittingsources 152 supported by a flexible PCB 151 that bounds one edge and oneface of the device 150. In certain embodiments, one or more lightemitting sources 152 may include a multi-emitter package arranged togenerate one or multiple peak wavelengths of light. The flexible PCB 151preferably includes a reflective surface arranged to reflect lighttoward a light-transmissive outer surface 159 of the device 150. Theflexible PCB 151 and the light emitting source(s) 152 are covered withan encapsulant material 154, which may include silicone. Between thelight-transmissive outer surface 159 and the encapsulant material 154,the device 150 further includes a diffusing and/or scattering layer 153.In certain embodiments, the diffusing and/or scattering layer 153 mayinclude a sheet of material; in other embodiments, the diffusing and/orscattering layer 153 may include particles applied in or on theencapsulant material 154. As illustrated, the device 150 may include athickness that is tapered with distance away from the light emittingsources 152. In certain embodiments, one or more light emitting sources152 may be arranged to produce one or both of ES increasing light and ESreleasing light.

FIG. 24 is a side cross-sectional schematic view of a portion of adevice 160 for delivering light energy to living mammalian tissue,wherein the device 160 is edge lit with one or more light emittingsources 162 supported by a flexible PCB 161 that bounds multiple edgesand a face of the device 160. In certain embodiments, one or more lightemitting sources 162 may include a multi-emitter package arranged togenerate one or multiple peak wavelengths of light. The flexible PCB 161preferably includes a reflective surface arranged to reflect lighttoward a light-transmissive outer surface 169 of the device 160. Theflexible PCB 161 and the light emitting source(s) 162 are covered withan encapsulant material 164, which may include silicone. Between thelight-transmissive outer surface 169 and the encapsulant material 164,the device 160 further includes a wavelength conversion material 163. Incertain embodiments, the wavelength conversion material 163 may includea sheet or layer of material; in other embodiments, the wavelengthconversion material 163 may include particles applied in or on theencapsulant material 164. In certain embodiments, one or more lightemitting sources 162 may be arranged to produce one or both of ESincreasing light and ES releasing light.

FIG. 25 is a side cross-sectional schematic view of a portion of adevice 170 for delivering light energy to living mammalian tissue,wherein the device 170 is edge lit with one or more light emittingsources 172 supported by a flexible PCB 171 that bounds one edge and oneface of the device 170. In certain embodiments, one or more lightemitting sources 172 may include a multi-emitter package arranged togenerate one or multiple peak wavelengths of light. The flexible PCB 171preferably includes a reflective surface arranged to reflect lighttoward a light-transmissive outer surface 179 of the device 170. Theflexible PCB 171 and the light emitting source(s) 172 are covered withan encapsulant material 174, which may include silicone. Between thelight-transmissive outer surface 179 and the encapsulant material 174,the device 170 further includes a wavelength conversion material 173. Incertain embodiments, the wavelength conversion material 173 may includea sheet or layer of material; in other embodiments, the wavelengthconversion material 173 may include particles applied in or on theencapsulant material 174. As illustrated, the device 170 may include athickness that is tapered with distance away from the light emittingsources 172. In certain embodiments, one or more light emitting sources172 may be arranged to produce one or both of ES increasing light and ESreleasing light.

FIG. 26 is a side cross-sectional schematic view of a portion of adevice 180 for delivering light energy to living mammalian tissue,wherein the device 180 is edge lit along multiple edges with multiplelight emitting sources 182 supported by a flexible PCB 181 having areflective surface arranged to reflect light toward a light-transmissiveouter surface 189 of the device 180. The flexible PCB 181 and lightemitting sources 182 are covered with an encapsulant material 184, and awavelength conversion material 183 is distributed in the encapsulantmaterial 184. In certain embodiments, one or more light emitting sources182 may include a multi-emitter package arranged to generate one ormultiple peak wavelengths of light. In certain embodiments, one or morelight emitting sources 182 may be arranged to produce one or both of ESincreasing light and ES releasing light.

FIG. 27 is a side cross-sectional schematic view of a portion of adevice 190 for delivering light energy to living mammalian tissue,wherein the device 190 is edge lit along multiple edges with multiplelight emitting sources 192 supported by a flexible PCB 191 having areflective surface arranged to reflect light toward a light-transmissiveouter surface 199 of the device 190. The device 190 further includesraised light extraction features 197 supported by the flexible PCB 191,with such features 197 serving to reflect laterally-transmitted lighttoward the outer surface 199. An encapsulant material 194 is providedover the flexible PCB 191, the light emitting sources 192, and the lightextraction features 197. In certain embodiments, one or more lightemitting sources 192 may include a multi-emitter package arranged togenerate one or multiple peak wavelengths of light. In certainembodiments, one or more light emitting sources 192 may be arranged toproduce one or both of ES increasing light and ES releasing light.

In certain embodiments, the light extraction features 197 may bedispensed, molded, layered, or painted on the flexible PCB 191. Incertain embodiments, different light extraction features 197 may includedifferent indices of refraction. In certain embodiments, different lightextraction features 197 may include different sizes and/or shapes. Incertain embodiments, light extraction features 197 may be uniformly ornon-uniformly distributed over the flexible PCB 191. In certainembodiments, light extraction features 197 may include tapered surfaces.In certain embodiments, different light extraction features 197 mayinclude one or more connected portions or surfaces. In certainembodiments, different light extraction features 197 may be discrete orspatially separated relative to one another. In certain embodiments,light extraction features 197 may be arranged in lines, rows, zig-zagshapes, or other patterns. In certain embodiments, one or morewavelength conversion materials may be arranged on or proximate to oneor more light extraction features 197.

FIG. 28 is a side cross-sectional schematic view of a portion of adevice 200 for delivering light energy to living mammalian tissue,wherein the device 200 is edge lit along multiple edges with multiplelight emitting sources 202 supported by a flexible PCB 201 having areflective surface arranged to reflect light toward a light-transmissiveouter surface 209 of the device 200. In certain embodiments, one or morelight emitting sources 202 may be arranged to produce one or both of ESincreasing light and ES releasing light. Encapsulant material layers204A, 204B are arranged above and below the flexible PCB 201 and overthe light emitting sources 202. Holes or perforations 205 are definedthrough the flexible PCB 201 and the encapsulant material layers 204A,204B. The holes or perforations 205 preferably allow passage of at leastone of air and exudate through the device 200.

Holes or perforations defined through a device (e.g., through a PCB andencapsulant layers) as described herein may include holes of variousshapes and configurations. Holes may be round, oval, rectangular,square, polygonal, or any other suitable axial shape. Cross-sectionalshapes of holes or perforations may be constant or non-constant.Cross-sectional shapes that may be employed according to certainembodiments are shown in FIGS. 29A-29C. FIG. 29A is a cross-sectionalview of a first exemplary hole 215A definable through an encapsulantlayer 214A of a device for delivering light energy to living mammaliantissue, the hole 215A having a diameter that is substantially constantwith depth and extending to an outer light transmissive surface 219A.FIG. 29B is a cross-sectional view of a second exemplary hole 215Bdefinable through an encapsulant layer 214B of a device for deliveringlight energy to living mammalian tissue, the hole 215B having a diameterthat increases with increasing depth and extending to an outer lighttransmissive surface 219B. FIG. 29C is a cross-sectional view of a thirdexemplary hole 215C definable through an encapsulant layer 214C of adevice for delivering light energy to living mammalian tissue, the hole215C having a diameter that decreases with increasing depth andextending to an outer light transmissive surface 219C.

In certain embodiments, perforations or holes may encompass at least 2%,at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, orat least 25% of a facial area of a device for delivering light energy toliving mammalian tissue as disclosed herein. In certain embodiments, oneor more of the preceding ranges may be bounded by an upper limit of nogreater than 10%, no greater than 15%, no greater than 20%, or nogreater than 30%. In certain embodiments, perforations or holes may beprovided with substantially uniform size and distribution, withsubstantially uniform distribution but non-uniform size, withnon-uniform size and non-uniform distribution, or any other desiredcombination of size and distribution patterns.

FIG. 30 is a top schematic view of at least a portion of a device 220for delivering light energy to living mammalian tissue, wherein thedevice 220 is edge lit along multiple edges with multiple light emittingsources 222 supported by a flexible PCB 221. The flexible PCB 221 ispreferably encapsulated on one or both sides with an encapsulantmaterial. Multiple holes or perforations 225 of substantially uniformsize and substantially uniform distribution are defined through theflexible PCB 221 and any associated encapsulant material layers. Theflexible PCB 221 preferably includes a reflective material arranged toreflect light toward a light transmissive outer surface 229 of thedevice 220. In certain embodiments, one or more light emitting sources222 may be arranged to produce one or both of ES increasing light and ESreleasing light.

FIG. 31 is a top schematic view of at least a portion of a device 230for delivering light energy to living mammalian tissue, wherein thedevice 230 is edge lit along multiple edges with multiple light emittingsources 232 supported by a flexible PCB 231. The flexible PCB 231 ispreferably encapsulated on one or both sides with an encapsulantmaterial. Multiple holes or perforations 235-1, 235-2 of differingsizes, but substantially uniform distribution, are defined through theflexible PCB 231 and any associated encapsulant material layers. Theflexible PCB 231 preferably includes a reflective material arranged toreflect light toward a light transmissive outer surface 239 of thedevice 230. In certain embodiments, one or more light emitting sources232 may be arranged to produce one or both of ES increasing light and ESreleasing light.

FIG. 32 is a top schematic view of at least a portion of a device 240for delivering light energy to living mammalian tissue, wherein thedevice 240 is edge lit along multiple edges with multiple light emittingsources 242 supported by a flexible PCB 241. The flexible PCB 241 ispreferably encapsulated on one or both sides with an encapsulantmaterial. The flexible PCB 241 preferably includes a reflective materialarranged to reflect light toward a light transmissive outer surface 249of the device 240. Multiple holes or perforations 245-1, 245-2 ofdifferent sizes are provided in one or more clusters 245A (e.g.,proximate to one or more light emitting sources 242) and defined throughthe flexible PCB 241 and any associated encapsulant material layers. Incertain embodiments, one or more light emitting sources 242 may bearranged to produce one or both of ES increasing light and ES releasinglight.

FIG. 33 is a top schematic view of at least a portion of a device 250for delivering light energy to living mammalian tissue, wherein thedevice 250 is edge lit along multiple edges with multiple light emittingsources 252 supported by a flexible PCB 251. The flexible PCB 251 ispreferably encapsulated on one or both sides with an encapsulantmaterial. The flexible PCB 251 preferably includes a reflective materialarranged to reflect light toward a light transmissive outer surface 259of the device 250. Multiple holes or perforations 255-1, 255-2 ofdifferent sizes and with a non-uniform (e.g., random) distribution aredefined through the flexible PCB 251 and any associated encapsulantmaterial layers. In certain embodiments, one or more light emittingsources 252 may be arranged to produce one or both of ES increasinglight and ES releasing light.

FIG. 34A is a top schematic view of at least a portion of a lightemitting device 260 for delivering light energy to living mammaliantissue and at least a portion of a battery/control module 270, whereinan elongated electrical cable 276 is associated with the battery/controlmodule 270 for connecting the battery/control module 270 to the lightemitting device 260. The light emitting device 260 is edge lit along oneedge with a light emitting region 261A supported by a flexible PCB 261.The flexible PCB 261 is preferably encapsulated on one or both sideswith an encapsulant material. The flexible PCB 261 preferably includes areflective material arranged to reflect light toward a lighttransmissive outer surface 269 of the device 260. Multiple holes orperforations 265 are defined through the flexible PCB 261 and anyassociated encapsulant material layers. One or more sensors 263 (e.g.,temperature sensors or any other types of sensors disclosed herein) arearranged in or on the flexible PCB 261. A socket 268 associated with thelight emitting device 260 is arranged to receive a plug 277 to which theelectrical cable 276 from the battery/control module 270 is attached.The battery / control module 270 includes a body 271, a battery 272, anda control board 273, which may include an emitter driver circuit and/orany suitable control, sensing, interface, data storage, and/orcommunication components as disclosed herein. The battery/control module270 may further include a port or other interface 278 to enablecommunication with an external device (e.g., laptop or tablet computer,a mobile phone, or another portable digital device) via wired orwireless means.

FIG. 34B is a top schematic view of at least a portion of a lightemitting device 280 for delivering light energy to living mammaliantissue and at least a portion of a battery/control module 290, whereinan elongated electrical cable 286 is associated with the light emittingdevice 280 for connecting the light emitting device 280 to thebattery/control module 290. The light emitting device 280 is edge litalong one edge with a light emitting region 281A supported by a flexiblePCB 281. The flexible PCB 281 is preferably encapsulated on one or bothsides with an encapsulant material. The flexible PCB 281 preferablyincludes a reflective material arranged to reflect light toward a lighttransmissive outer surface 289 of the device 280. Multiple holes orperforations 285 are defined through the flexible PCB 281 and anyassociated encapsulant material layers. One or more sensors 283 (e.g.,temperature sensors or any other types of sensors disclosed herein) arearranged in or on the flexible PCB 281. A socket 298 associated with thebattery/control module 290 is arranged to receive a plug 287 to whichthe electrical cable 286 from the light emitting device 280 is attached.The battery/control module 290 includes a body 291, a battery 292, and acontrol board 293, which may include an emitter driver circuit and/orany suitable control, sensing, interface, data storage, and/orcommunication components as disclosed herein. The light emitting device280 may further include a port or other interface 288 to enablecommunication with an external device (e.g., laptop or tablet computer,a mobile phone, or another portable digital device) via wired orwireless means.

FIG. 35 is a top schematic view of at least a portion of a lightemitting device 300 for delivering light energy to living mammaliantissue and being connected via an electrical cord 316 to abattery/control module 310, wherein the light emitting device 300includes multiple light emitters 302 supported by a flexible PCB 301,multiple holes or perforations 305, and multiple sensors 303A-303C. Theflexible PCB 301 is preferably encapsulated on one or both sides with anencapsulant material. The flexible PCB 301 preferably includes areflective material arranged to reflect light toward a lighttransmissive outer surface 309 of the device 300. Multiple holes orperforations 305 are defined through the flexible PCB 301 and anyassociated encapsulant material layers. Multiple sensors 303A-303C arearranged in or on the flexible PCB 301. In certain embodiments, thesensors 303A-303C may differ in type from one another. In certainembodiments, the sensors 303A-303C may include one or more lightemitters and photodiodes to illuminate a wound site with one or moreselected wavelengths (e.g., green light) to detect blood flow in orproximate to a wound site to provide photoplethsmyography data. Thesensors 303A-303C may alternatively or additionally be arranged todetect blood pressure, bandage or dressing covering pressure, heartrate, temperature, presence or concentration of chemical or biologicalspecies (e.g., in wound exudate), or other conditions. A socket 308associated with the light emitting device 300 is arranged to receive aplug 317 to which the electrical cord 316 from the battery/controlmodule 310 is attached. The battery/control module 310 includes a body311, a battery 312, and a control board 313, which may include anemitter driver circuit and/or any suitable control, sensing, interface,data storage, and/or communication components as disclosed herein. Thebattery/control module 310 may further include a port or other interface318 to enable communication with an external device (e.g., laptop ortablet computer, a mobile phone, or another portable digital device) viawired or wireless means.

FIGS. 36A-36C illustrate different pulse profiles that may be used withdevices and methods according to the present disclosure. FIG. 36A is aplot of intensity versus time embodying a first exemplary illuminationcycle that may be used with at least one emitter of a light emittingdevice for delivering light energy to living mammalian tissue asdisclosed herein. As shown in FIG. 36A, a series of discrete pulses ofsubstantially equal intensity may be provided during at least one timewindow or a portion thereof. FIG. 36B is a plot of intensity versus timeembodying a second exemplary illumination cycle that may be used with atleast one emitter of a light emitting device disclosed herein. As shownin FIG. 36B, intensity may be reduced from a maximum (or high) value toa reduced but non-zero value during at least one time window. FIG. 36Cis a plot of intensity versus time embodying a third exemplaryillumination cycle that may be used with at least one emitter of a lightemitting device disclosed herein. As shown in FIG. 36C, intensity may besteadily reduced from a maximum (or high) value to sequentially reducedvalues over time. Other pulse profiles may be used according to certainembodiments.

FIG. 37 is an exploded view of a light emitting device 405 embodied in awearable cap for delivering light energy to a scalp of a patient. Thedevice 405 includes multiple light emitters and standoffs supported by aflexible PCB 410 including multiple interconnected panels 412A-412Farranged in a concave configuration. A concave shaping member 430(including a central frame 431, ribs 432A-432D, and curved panels434A-434D) is configured to receive the flexible PCB 410. The ribs432A-432D and curved panels 434A-434D project generally outwardly anddownwardly from the central frame 431. Gaps are provided betweenportions of adjacent ribs 432A-432D and curved panels 434A-434D toaccommodate outward expansion and inward contraction, and to enabletransfer of heat and/or fluid (e.g., evaporation of sweat). A fabriccovering member 460 is configured to cover the concave shaping member430 and the flexible PCB 410 contained therein. A battery 450 and abattery holder 451 are arranged between the flexible PCB 410 and theconcave shaping member 430. An electronics housing 440 is arranged to bereceived within an opening 431A defined in the central frame 431 of theconcave shaping member 430. Pivotal coupling elements 441A, 451A arearranged to pivotally couple the battery holder 451 to the electronicshousing 440. An electronics board 441 is insertable into the electronicshousing 440, which is enclosed with a cover 442. Arranged on theelectronics board 441 are a cycle counter 443, a control button 444, acharging/data port 445, and a status lamp 446. The various elementsassociated with the electronics housing 440 and the electronics board441 may be referred to generally as a “control module.” Windows 442Adefined in the cover 442 provide access to the cycle counter 443, thecontrol button 444, the charging/data port 445, and the status lamp 446.The fabric covering element 460 includes a fabric body 461 and multipleinternal pockets 462A-462D arranged to receive portions of the ribs432A-432D. An opening 468 at the top of the fabric covering element 460is arranged to receive the cover 442.

FIG. 38 is a bottom plan view of the flexible PCB 410 including lightemitters 420 and standoffs 425 arranged thereon. The PCB 410 includes apolyimide substrate 411, an inner surface 411A, and an outer surface 411B (shown in FIG. 37). In one embodiment, the light emitters 420 includea total of 280 light emitting diodes arranged as 56 strings of 5 LEDs,with a string voltage of 11V, a current limit of 5 mA, and a powerconsumption of 3.08 watts. FIG. 38 illustrates 36 standoffs 425extending from the inner surface 411A of the flexible PCB 410. Theflexible PCB 410 includes six interconnected panels 412A-412F, with thepanels 412A-412F being connected to one another via narrowed tab regions413B-413F. Gaps 414A-414F are provided between the various panels412A-412F, with such gaps 414A-414F (which are extended proximate to thenarrowed tab regions 413B-413F) being useful to permit transport of heatand/or fluid (e.g., evaporation of sweat) between the panels 412A-412F.As shown in FIG. 38, holes 415A, 415B are defined through the substrate411 to receive fasteners (not shown) for joining the flexible PCB 410 tocorresponding holes (not shown) defined in the electronics housing 440.A further opening 415C may be provided for sensor communication betweena proximity sensor (e.g., photosensor) and the interior of the flexiblePCB 410 when the flexible PCB 410 is shaped into a concaveconfiguration.

FIG. 39 is a front elevation view of the assembled light emitting device405 embodied in the wearable cap of FIG. 37 superimposed over a modeledhuman head. As shown in FIG. 39, the device 405 is embodied in a capwith a lower edge between a user's forehead and hairline, and above auser's ears.

FIG. 40 is a schematic diagram showing interconnections betweencomponents of a light emitting device for delivering light energy totissue of a patient according to one embodiment. A microcontroller 502is arranged to receive power from a battery 522 (nominally 3.7V) via a5V voltage boost circuit 512. The microcontroller may be arranged tocontrol a charging integrated circuit 514 arranged between a microUSBconnector 516 and the battery 522, wherein the microUSB connector 516may be used to receive current for charging the battery 522. In certainembodiments, the microUSB connector 516 may also be used forcommunicating data and/or instructions to or from the microcontroller502 and/or an associated memory. The microcontroller 502 is alsoarranged to control a 12V boost circuit 518 for increasing voltage toone or more LED arrays 520. The microcontroller 502 further controls oneor more LED driver circuits 510 arranged to drive the LED array(s) 520.The microcontroller 502 is also arranged to receive inputs from a userinput button 504, a temperature sensor 524, and a proximity sensor 526(which includes an infrared LED 528). The microcontroller 502 is furtherarranged to provide output signals to a LCD display 506 and a buzzer508. Certain components are located off-board relative to a controllerPCB, as indicated by the vertical dashed line in FIG. 40. In operationof the light emitting device, a user may depress the button 504 to startoperation. If the proximity sensor 526 detects that the device has beenplaced in suitable proximity to desired tissue, then the microcontroller502 may trigger the LED driver circuit(s) 510 to energize the LEDarray(s) 520. Temperature during operation is monitored with thetemperature sensor 524. If an excess temperature condition is detected,then the microcontroller 502 may take appropriate action to reducecurrent supplied by the LED driver circuit(s) 510 to the LED array(s)520. Operation may continue until a timer (e.g., internal to themicrocontroller 502) causes operation to terminate automatically. One ormore indicator LEDs (not shown) may provide a visible signal indicativeof charging status of the battery 522. Audible signals for commencementand termination of operation may be provided by the buzzer 508 or asuitable speaker. Information relating to usage cycles, usage time, orany other suitable parameter may be displayed by the LCD display 506.

FIG. 41 is a schematic diagram depicting an interface between hardwaredrivers, functional components, and a software application suitable foroperating a light emitting device according to FIG. 40. Applicationexecutive functions 503, including timers and counters 507, may beperformed with one or more integrated circuits (such as themicrocontroller 502 illustrated in FIG. 40). Hardware drivers 505 may beused to interface with various input and output elements, such as theLED array(s) 520, the speaker or buzzer 508, the LCD display 506, thetemperature sensor 524, the push button 504, the indicator LEDs 509, andthe optical sensor (proximity sensor) 526.

FIG. 42 is a schematic elevation view of at least a portion of a lightemitting device 600 for delivering light energy to tissue in an internalcavity (e.g., body cavity) of a patient according to one embodiment. Incertain embodiments, a body cavity may comprise a vaginal cavity, anoral cavity, or an esophageal cavity. If used in an oral or esophagealcavity, one or more unobstructed channels or tubes (not shown) may beprovided in, on, or through the device 600 to avoid interruption withpatient breathing. The device 600 includes a body 601 that may be rigid,semi-rigid, or articulated. A treatment head 603 has arranged therein orthereon one or more light emitters 605, which are preferablyencapsulated in silicone or another suitable light transmissivematerial. In certain embodiments, the one or more light emitters 605 maybe arranged to produce ES increasing light and ES releasing light forimpingement on tissue located within an internal cavity of a patient totrigger release of NO. In certain embodiments, the light emitters may beexternal to the body 601, and light emissions of the light emitters maybe extracted at features that are arranged on the end of the body 601(e.g., in or along treatment head 603), and light may exit the treatmenthead 603 at apertures or positions corresponding to element number 605.

FIG. 43A is a schematic elevation view of at least a portion of a lightemitting device 610 including a concave light emitting surface 614including one or more light emitters 615 for delivering light energy tocervical tissue of a patient according to one embodiment. The device 610includes a body 611 that may be rigid, semi-rigid, or articulated. Ajoint 612 may be arranged between the body 611 and a treatment head 613.The treatment head 613 has arranged therein or thereon the one or morelight emitters 615, which are preferably encapsulated in silicone oranother suitable light transmissive material. In certain embodiments,the one or more light emitters 615 may be configured to generateemissions suitable for neutralizing pathogens such as human papillomavirus (HPV) present on cervical tissue. In certain embodiments, the oneor more light emitters 615 may be arranged to produce ES increasinglight and ES releasing light for impingement on tissue located within aninternal cavity of a patient to trigger release of NO.

FIG. 43B illustrates the device 610 of FIG. 43A inserted into a vaginalcavity 650 to deliver light energy to cervical tissue 655 of a patientproximate to a cervical opening 656. The concave light emitting surface614 may be configured to approximately match a convex profile of thecervical tissue 655.

FIG. 44A is a schematic elevation view of at least a portion of a lightemitting device 620 including a light emitting surface 624 with aprotruding probe portion 626 for delivering light energy to cervicaltissue of a patient according to another embodiment. The probe portion626 includes light emitters and is arranged to deliver light energy intoa cervical opening. The device 620 includes a body 621 that may berigid, semi-rigid, or articulated. A joint 622 may be arranged betweenthe body 621 and a treatment head 623. The treatment head 623 hasarranged therein or thereon one or more light emitters 625, which arepreferably encapsulated in silicone or another suitable lighttransmissive material. The treatment head 623 may include the lightemitting surface 624, which may optionally be convex to cast a wideroutput beam. In certain embodiments, the one or more light emitters 625may be configured to generate emissions suitable for neutralizingpathogens such as human papilloma virus (HPV) present on cervicaltissue. In certain embodiments, the one or more light emitters 625 maybe arranged to produce ES increasing light and ES releasing light forimpingement on tissue located within an internal cavity of a patient totrigger release of NO.

FIG. 44B illustrates the device 620 of FIG. 44A inserted into a vaginalcavity 650 to deliver light energy to cervical tissue 655 of a patientproximate and within to a cervical opening 656. The primary lightemitting surface 624 may be arranged to impinge light on cervical tissuebounding the vaginal cavity 650, whereas the probe portion 626 may beinserted into the cervical opening 656 to deliver additional lightenergy therein to increase the amount of cervical tissue subject toreceipt of light energy for addressing one or more conditions includingpathogen (e.g., HPV) neutralization.

To investigate whether NO may be photomodulated in at least certaintypes of cells for extended periods (e.g., hours) and to evaluatepotential toxicity of photomodulation, Applicant performed variousexperiments on two types of cells—namely, keratinocytes and fibroblasts.

Referring to FIGS. 45-48, isolated keratinocytes were exposed to 420 nmlight to achieve doses of 0, 1, 5, and 50 J/cm². Fluence of light wasfound to determine efficacy of NO modulation as well as cytotoxicity. Asshown in FIG. 45, cell viability over periods from 0 to 24 hours fromlight exposure was unaffected by doses of 0, 1, and 5 J/cm², but lightexposure at 50 J/cm² resulted in a substantial drop in cell viability,declining to a value below 20% within 24 hours after irradiation.

Referring to FIGS. 46 and 47, the amount of NOS enzymes (namely, iNOS inFIG. 46, and nNOS in FIG. 47) expressed in the keratinocyte cells wasquantified at intervals of 0 hours (immediately), 1 hour, 4 hours, and 8hours after irradiation ended. The number of cells exhibiting iNOS andnNOS increased with increasing irradiation. In FIG. 46, the percentageof cells expressing iNOS generally remained the same or decreased 1 hourafter light exposure; the percentage of cells expressing iNOS increasedfor doses of 1 and 50 J/cm² at a time 4 hours after light exposure, andthe percentage of cells expressing iNOS remained elevated only for thedose of 50 J/cm² at a time 24 hours after light exposure. In FIG. 47,the percentage of cells expressing nNOS generally increased for alldoses of 0, 1, 5, and 50 J/cm² at a time 1 hour after light exposure,the percentage of cells expressing nNOS remained elevated only for thedose of 50 J/cm² at time periods of 4 hours and 8 hours after lightexposure. FIGS. 46 and 47 show the capability of generated nitric oxidesynthases with photomodulation.

Referring to FIG. 48, intracellular NO was measured with4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate (DAF-FMDiacetate). The number of cells exhibiting intracellular NO increasedwith increasing irradiation. Intracellular NO was measured immediatelyafter light exposure as well as 4 and 8 hours after exposure. FIG. 48shows that NO is released for greater than 4 hours after irradiation,thereby suggesting enzymatic NO generation.

Turning to FIGS. 49-52, isolated fibroblasts were exposed to 420 nmlight to achieve doses of 0, 5, 25, and 50 J/cm². Fluence of light wasfound to determine efficacy of NO modulation as well as cytotoxicity. Asshown in FIG. 49, cell viability over periods from 0 to 24 hours fromlight exposure was substantially unaffected by doses of 0, 5, 25, and 50J/cm².

Referring to FIGS. 50 and 51, the amount of NOS enzymes (namely, iNOS inFIG. 50, and eNOS in FIG. 51) expressed in the fibroblast cells wasquantified at intervals of 0 hours (immediately), 1 hour, and 6 hoursafter irradiation ended. In both figures, the number of cells exhibitingiNOS or eNOS generally increased with increasing irradiation. In FIG.50, the percentage of cells expressing iNOS was particularly elevatedfor the dose of 50 J/cm² at a time period of 6 hours after irradiation,thereby suggesting enzymatic NO generation. Referring to FIG. 51, thepercentage of cells expressing eNOS remained generally elevated at atime period of 6 hours after irradiation, but the dose of 50 J/cm² wasparticularly elevated at this time period.

Referring to FIG. 52, intracellular NO was measured with4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate (DAF-FMDiacetate). The number of cells exhibiting intracellular NO increasedwith increasing irradiation. Intracellular NO was measured immediatelyafter light exposure as well as 1 and 6 hours after exposure. FIG. 52shows that NO is released for greater than 4 hours after irradiation,thereby suggesting enzymatic NO generation. The percentage of cells withintracellular NO remained elevated at 1 hour and 6 hours afterirradiation for the dose of 50 J/cm², but was particularly elevated at 6hours for 50 J/cm².

Taken in combination, FIGS. 49-52 demonstrate the capability ofgenerating nitric oxide synthases and NO using 420 nm light for 6 hourspost irradiation without associated toxicity.

Efficacy of the liberation of nitric oxide from protein complexes (bybreaking nitroso or nitrosyl bonds) depends on the wavelength of lightused. Different types of bonds (e.g., RSNO, RNNO, and metal-NO) mayrequire different light wavelength and light irradiation values toeffectuate release of nitric oxide. To investigate whether certain lightwavelengths and light irradiation values may be more effective thanothers at releasing different endogenous stores of NO (i.e., to serve asES releasing light), Applicant performed various experiments withhemoglobin-NO, S-nitrosoglutathione (GSNO), albumin-NO, cytochrome c-NO,cytochrome c-oxidase-NO, and mitochondria-NO. Details of theseexperiments are described hereinafter in connection with FIGS. 53 to 64.

FIG. 53 is a plot of NO release rate (PPB/s) versus irradiance (J/cm²)from hemoglobin-NO for nine (9) different wavelengths of incoherentlight ranging from 410 nm to 850 nm. Nitric oxide was added tohemoglobin by reacting proline-NONOate with hemoglobin in PBS (pH 6.5)under anaerobic conditions and in the dark. After 45 minutes ofreaction, the NO release was measured as a function of irradiation usinga chemiluminescence detector. As shown, all wavelengths resulted inrelease of NO (at a roughly constant rate for all irradiance valuesgreater than about 2 J/cm²), but the release rate was highest for 420 nmlight, second highest for 410 nm light, and lowest for longerwavelengths (e.g., 850 nm light). Referring to FIG. 54, total NOreleased from hemoglobin was quantified by integrating the data on NOrelease rate of FIG. 53. A linear relationship is observed for eachwavelength, with higher irradiance values resulting in higher total NOrelease. The highest amount of total NO release was achieved with 420 nmlight, the second highest amount was achieved with 410 nm light, and thelowest amount of total NO release was achieved with 597 nm light.Notably, FIG. 54 omits data for 660 nm light and 850 nm light.

FIG. 55 is a plot of NO release rate (PPB/s) versus irradiance (J/cm2)from S-nitrosoglutathione (GSNO) for ten (10) different wavelengths ofincoherent light ranging from 410 nm to 850 nm. The NO-release fromS-nitrosoglutathione was measured in PBS (pH 6.5), at room temperatureas a function of irradiation via chemiluminescent detection. As shown,all wavelengths resulted in some release of NO, but the release rate washighest for the shortest wavelength (410 nm) light and lowest for thelongest wavelength (850 nm) light. Referring to FIG. 56, total NOreleased from hemoglobin was quantified by integrating the data on NOrelease rate of FIG. 55. The highest amounts of total NO release wereachieved with 410 nm and 420 nm light, and the lowest amount of total NOrelease was achieved with 850 nm light.

FIG. 57 is a plot of NO release rate (PPB/s) versus irradiance (J/cm²)from albumin-NO for nine (9) different wavelengths of incoherent lightranging from 420 nm to 850 nm. Nitric oxide was added to bovine serumalbumin by reacting with proline-NONOate in PBS (pH 6.5) under anaerobicconditions and in the dark. After 45 minutes of reaction, the NO releasewas measured as a function of irradiation using a chemiluminescencedetector. As shown, the highest NO release rate was achieved for thewavelength of 448 nm, and the second and third highest NO release rateswere achieved for wavelengths of 420 nm and 470 nm, respectively, witheach of the foregoing three wavelengths causing an initial spike orincrease in NO release rate followed by a lower release rate. Referringto FIG. 58, total NO released from albumin-NO was quantified byintegrating the data on NO release rate of FIG. 57. Similar amounts oftotal NO release were achieved for 420 nm light, 448 nm light, and 470nm light. An intermediate amount of total NO release was achieved for505 nm light. Relatively little total NO release was achieved for lightof other wavelengths.

FIG. 59 is a plot of NO release rate (PPB/s) versus irradiance (J/cm²)from cytochrome c-NO for ten (10) different wavelengths of incoherentlight ranging from 410 nm to 850 nm. Nitric oxide was added tocytochrome c by reacting proline-NONOate in PBS (pH 6.5) under anaerobicconditions and in the dark. After 45 minutes of reaction, the NO releasewas measured as a function of irradiation using a chemiluminescencedetector. As shown, the highest four NO release rates were achieved for420 nm light, 410 nm light, 448 nm light, and 470 nm light,respectively, with each exhibiting a peak release rate near anirradiance value of about 2 J/cm², while all wavelengths exhibiting areduced or negligible NO release rate as irradiance values approached 20J/cm². Referring to FIG. 60, total NO released from cytochrome c-NO wasquantified by integrating the data on NO release rate of FIG. 59. Asshown, the highest four amounts of total NO release were achieved for420 nm light, 410 nm light, 448 nm light, and 470 nm light,respectively.

FIG. 61 is a plot of NO release rate (PPB/s) versus irradiance (J/cm²)from cytochrome c-oxidase NO for ten (10) different wavelengths ofincoherent light ranging from 410 nm to 850 nm. Nitric oxide was addedto cytochrome c oxidase by reacting with proline-NONOate in PBS (pH 6.5)under anaerobic conditions and in the dark. After 45 minutes ofreaction, the NO release was measured as a function of irradiation usinga chemiluminescence detector. As shown, the highest four NO releaserates were achieved for 410 nm light, 420 nm light, 448 nm light, and470 nm light, respectively. For 410 nm light, NO release rate generallyincreased with increasing irradiance, whereas for other wavelengths, atleast a local peak of NO release rate was achieved for irradiance valuesof around 1 to 2 J/cm², followed by an increase in NO release rate withincreasing irradiance for 420 nm light, 448 nm light, and 470 nm light,but higher wavelengths of light resulted in decreased NO release ratewith increasing irradiance. Referring to FIG. 62, total NO released fromcytochrome c-oxidase-NO was quantified by integrating the data on NOrelease rate of FIG. 61. The highest three amounts of total NO releasewere achieved for 410 nm light, 420 nm light, and 448 nm light,respectively, with greater slopes for shorter wavelengths.

FIG. 63 is a plot of NO release rate (PPB/s) versus irradiance (J/cm²)from mitochondria-NO for ten (10) different wavelengths of incoherentlight ranging from 410 nm to 850 nm. Nitric oxide was added tomitochondria isolated from bovine heart by reacting it withS-nitrosoglutatione in PBS (pH 6.5) under anaerobic conditions and inthe dark. After 45 minutes of reaction, the NO release was measured as afunction of irradiation using a chemiluminescence detector. As shown,the highest four NO release rates were achieved for 410 nm light, 420 nmlight, 448 nm light, and 470 nm light, respectively. For eachwavelength, a peak of NO release rate was achieved for irradiance valuesin a range of from about 2 to 4 J/cm², followed by a decrease in NOrelease rate with increasing irradiance. Referring to FIG. 64, total NOreleased from mitochondria-NO was quantified by integrating the data onNO release rate of FIG. 63. The highest four amounts of total NO releasewere achieved for 410 nm light, 420 nm light, 448 nm light, and 470 nmlight, respectively.

The preceding FIGS. 53 to 64 show that different types of bonds (e.g.,RSNO, RNNO, and metal-NO) may require different light wavelengths and/orlight irradiation values to effectuate release of nitric oxide. Based onthe data represented in FIGS. 53 to 64, an important wavelength ofinterest is 420 nm, since this wavelength represents perhaps the closestsafe wavelength to the ultraviolet range (since substantially allincoherent emissions having a peak wavelength of 420 nm, includingportions tailing above and below this peak value, remain well above the400 nm UV threshold), exhibits a demonstrated high (or highest) NOrelease from a wide range of proteins (Hemoglobin-NO,S-Nitrosglutathione (GSNO), Albumin-NO, Cytochrome c-NO, Cytochrome coxidase-NO, and Mitochondria-NO), and appears to lead to enzymaticgeneration of NO. A secondary wavelength of interest is 530 nm, since itappears to be more effective than longer wavelength red at triggering NOrelease from GSNO. These conclusions contradict various findings in theart (e.g., by Karu, T, Handbook of Laser Wavelengths, Chapter 48,“Low-Power Laser Therapy”, pp. 48-1 to 48-25 (2003); by Ball, K., etal., “Low intensity light stimulates nitrite-dependent nitric oxidantsynthesis but not oxygen consumption by cytochrome c oxidase:implications for phototherapy,” Journal of Photochemistry andPhotobiology B: Biology 102 (2011) 182-191; and by Hamblin, M., “TheRole of Nitric Oxide in Low Level Light Therapy,” Proc. of SPIE Vol.6846, 684602, (2008)) that red light including wavelengths in a range offrom 605 nm to 820 nm may be particularly suitable for releasing NO fromheme groups of CCO, for release of NO from CCO generally, and forincreased ATP synthesis.

Based on the findings that short wavelength blue light is effective forenhancing endogenous stores of nitric oxide and/or triggering nitricoxide release, one aspect of the disclosure relates to a method ofmodulating nitric oxide in living mammalian tissue, the methodcomprising: impinging light on the tissue, wherein the light impinged onthe tissue comprises incoherent light emissions including a first peakwavelength in a range of from 410 nm to 440 nm and a first radiant flux,and wherein the first peak wavelength and the first radiant flux areselected to stimulate at least one of (i) enzymatic generation of nitricoxide to increase endogenous stores of nitric oxide or (ii) release ofnitric oxide from endogenous stores of nitric oxide; wherein the lightimpinged on the tissue is substantially devoid of light emissions havinga peak wavelength in a range of from 600 nm to 900 nm (e.g.,encompassing red visible light as well as a portion of the infraredrange). An absence of red and/or infrared light contradicts variousreferences describing the desirability of red and/or infrared light asprimary wavelengths for skin penetration and to provide phototherapeuticbenefit.

In certain embodiments, the light impinged on the tissue is devoid ofemissions of any wavelength conversion material (e.g., a phosphor, aquantum dot, or another lumiphoric material) stimulated by incoherentlight emissions having a peak wavelength in a range of from 410 nm to440 nm. In certain embodiments, the tissue on which light is impinged isdevoid of an applied or received photosensitive therapeutic compound oragent (e.g., a pharmaceutical composition or the like, which may beadministered topically, orally, or via injection). In certainembodiments, at least 65%, at least 75%, at least 80%, at least 85%, orat least 95% of a fluence of light impinged on the tissue consists ofthe incoherent light emissions including a first peak wavelength in arange of from 410 to 440 nm. In certain embodiments, the light impingedon the tissue is substantially devoid of light emissions having a peakwavelength in a range of from 441 nm to 490 nm. In certain embodiments,the incoherent light emissions including a first peak wavelength in arange of from 410 nm to 440 nm are provided as a plurality of discretepulses.

In certain embodiments, the light impinged on the tissue furthercomprises incoherent light emissions including a second peak wavelengthin a range of from 500 nm to 540 nm. This is consistent with Applicant'sfinding that light having a peak wavelength of 530 nm appears to be moreeffective than certain other wavelengths (including longer wavelengthred) at triggering NO release from GSNO. In certain embodiments, theincoherent light emissions including a first peak wavelength in a rangeof from 410 nm to 440 nm are impinged on the tissue during a first timewindow, the incoherent light emissions including a second peakwavelength in a range of from 500 nm to 540 nm are impinged on thetissue during a second time window, and at least a portion of the secondtime window is non-overlapping with the first time window.

In certain embodiments, the first peak wavelength and the first radiantflux are selected to stimulate enzymatic generation of nitric oxide toincrease endogenous stores of nitric oxide. In certain embodiments, thefirst peak wavelength and the first radiant flux are selected to releasenitric oxide from the endogenous stores of nitric oxide.

In certain embodiments, the tissue comprises at least one of epithelialtissue, mucosal tissue, connective tissue, muscle tissue, or cervicaltissue. In certain embodiments, the tissue comprises dermal tissue. Incertain embodiments, a method further comprises sensing a temperaturecondition on or proximate to (a) a therapeutic device arranged toimpinge light on the tissue, or (b) the tissue; generating at least onesignal indicative of the temperature condition; and controllingimpingement of light on the tissue responsive to the at least onesignal. In certain embodiments, the light impinged on the tissuecomprises a fluence in a range of from about 0.5 J/cm²to about 100J/cm², or from about 2 J/cm² to about 80 J/cm², or from about 5 J/cm² toabout 50 J/cm².

In another aspect, the disclosure relates to a device for modulatingnitric oxide in living mammalian tissue, the device comprising: anambient light blocking element; and at least one first light emittingelement positioned between the ambient light blocking element and thetissue, wherein the at least one first light emitting element isconfigured to impinge incoherent light on the tissue, said incoherentlight having a first peak wavelength and a first radiant flux, whereinthe first peak wavelength and the first radiant flux are selected tostimulate at least one of (i) enzymatic generation of nitric oxide toincrease endogenous stores of nitric oxide or (ii) release of nitricoxide from endogenous stores of nitric oxide; wherein the device issubstantially devoid of any light emitting element configured to impingeon the tissue light having a peak wavelength in a range of from 600 nmto 900 nm.

In certain embodiments, the device is substantially devoid of any lightemitting element configured to impinge light having a peak wavelength ina range of from 441 nm to 490 nm on the tissue. In certain embodiments,the device is devoid of any wavelength conversion material configured tobe stimulated by the at least one first light emitting element. Incertain embodiments, the device further comprises a flexible substratesupporting the at least one first light emitting element. In certainembodiments, the device is configured to conform to the tissue with alight-transmissive material arranged in contact with the tissue. Incertain embodiments, the light impinged on the tissue is substantiallydevoid of light emissions having a peak wavelength in a range of from441 nm to 490 nm. In certain embodiments, the device further comprisesdriver circuitry configured to generate the incoherent light emissionsincluding the first peak wavelength, wherein the first peak wavelengthis in a range of from 410 nm to 440 nm, and said incoherent lightemissions comprise a plurality of discrete pulses.

In certain embodiments, the device further comprises at least one secondlight emitting element configured to impinge incoherent light on thetissue, said incoherent light having a second peak wavelength and asecond radiant flux, wherein the second peak wavelength is in a range offrom 500 nm to 540 nm. In certain embodiments, the device is configuredto impinge incoherent light emissions including the first peakwavelength during a first time window, wherein the first peak wavelengthis in a range of from 410 nm to 440 nm, and being configured to impingeincoherent light emissions including the second peak wavelength in arange of from 500 nm to 530 nm during a second time window, wherein atleast a portion of the second time window is non-overlapping with thefirst time window. In certain embodiments, the device further comprisesa probe configured for insertion into a mammalian body cavity or opening(e.g., incision) defined in a mammalian body, wherein the at least onefirst light emitting element is supported by the probe.

FIG. 65 is a perspective view illustration of a cross-section of dermisand epidermis layers of human skin showing various types of cellscontaining nitric oxide synthases or enzymes. Such image is reproducedfrom Cals-Grierson, M. M and Ormerod, A.D. Nitric Oxide 10 (2004)179-193. As shown, the epidermis (extending from the stratum corneum toand including the basal layer) includes keratinocytes, Langerhans cells,and melanocytes, whereas the dermis (under the basal layer) includesfibroblasts and microvasculature. Different NOS enzymes occur indifferent layers of the skin. nNOS (or NOS1) is present inkeratinocytes; eNOS (or NOS3) is present in fibroblasts, melanocytes,and the endothelium; and iNOS (or NOS2) is present throughout. Both nNOSand eNOS are calcium dependent enzymes. iNOS is inducible and thereforeincreases in response to the immune system.

FIG. 66 is a related art cross-sectional illustration of human skin witha superimposed representation of depth penetration of coherent (e.g.,laser) light of eight different wavelengths ranging from 420 nm to 755nm. Such image is sourced fromwww.spectrumsciencebeauty.com.au/2014/09/16/ipl-hair-removal/#prettyPhoto/0/.FIG. 66 shows a single hair follicle (below the value of “640 nm”, at adepth of between 2 and 3 mm). As shown, the conclusion in the art isthat blue light (e.g., 420 nm, 480 nm) is incapable of penetrating humanskin to a sufficient depth to reach a hair follicle.

Applicant performed various experiments to contradict thisconclusion—instead confirming that coherent blue light is capable ofpenetrating human skin to a depth sufficient to reach hair follicles.Irradiance transmitted through full thickness skin was measured as afunction of wavelength for laser and LED light sources. Light sourceswere matched to have equivalent irradiance as measured by a commonphotodiode. Wavelength was also matched between laser and LED lightsources. FIGS. 67A, 68A, and 69A embody upper perspective viewphotographs of transmittance of incoherent (LED) light and coherent(laser) light through Caucasian (Fitzpatrick Skin Type II) human skinsamples, with the respective figures separately corresponding to red(660 nm peak wavelength), green (530 nm peak wavelength), and blue (420nm peak wavelength) sources. Human skin samples of different thicknesses(1.3 mm and 2.5 mm) were used in each instance. FIGS. 67B, 68B, and 69Bembody plots of light transmittance percentage as a function of skinthickness (mm) for transmittance of incoherent (LED) light and coherent(laser) light through the human skin samples of two differentthicknesses. In each of FIGS. 67B, 68B, and 69B, a significantly greaterpercentage of incoherent (LED) light was transmitted through skin thancoherent (laser) light. Notably, referring to FIG. 69B, nearly 40% ofthe incoherent (420 nm peak) blue light was transmitted through aCaucasian skin sample having a thickness of 2.5 mm, whereas a low singledigit percentage of coherent blue light was transmitted through the samesample.

To determine whether red, green, and blue coherent and incoherent lightcan penetrate skin of racially diverse types, experiments were performedusing the apparatuses of FIGS. 67A, 68A, and 69A using human skinsamples of three different thicknesses for each of two differentpigmentations (i.e., African American skin of Fitzpatrick Skin Type V,and Caucasian skin of Fitzpatrick Skin Type II). Results of theseexperiments for red (660 nm peak wavelength), green (530 nm peakwavelength), and blue (420 nm peak wavelength) sources are shown inFIGS. 70 to 72, respectively. As shown, despite the different skinpigmentation, the samples of African American skin of Fitzpatrick SkinType V and samples of Caucasian skin of Fitzpatrick Skin Type II skinsamples performed similarly with respect to light transmittanceproperties. As shown in FIG. 70, red incoherent (LED) light wastransmitted through each sample at more than twice the percentage of redcoherent (laser) light. As shown in FIGS. 71 and 72, green and blueincoherent (LED) light were transmitted through each sample at more thantwice the percentage of green and blue coherent (laser) light,respectively. Conclusions to be gleaned from the foregoing experimentsare that LEDs appear to be at least as effective as lasers (forwavelengths of 420-660 nm) at penetrating skin of different types; andthat a high percentage of blue LED light is capable of penetratingCaucasian and African American skin at depths of 2.5 mm or more.

In certain embodiments, methods and devices disclosed herein may be usedto enhance nitric oxide production and/or release to provide a hair losssolution (e.g., for treating androgenic alopecia and/or similarconditions). Hair loss is caused by an increase in DHT produced by theenzyme 5α-reductase. In particular, 5α-reductase reacts withtestosterone and NADPH to produce dihydrotestosterone (DHT), which leadsto shrinkage of hair follicles and hair loss. Applicant performedexperiments to determine whether nitric oxide inhibits 5α-reductase, tothereby provide a potential for decreasing DHT concentration in thescalp and inhibit (or reverse) hair loss. S-Nitrosoglutathione (GSNO)was used as a NO donor. Nitric oxide is released from GSNO by NADPH,which is a necessary cofactor for the 5α-reductase enzyme.

FIG. 73 is a plot of percentage of DHT remaining as a function ofNO-donor concentration (mM) for six values ranging from 0 to 50 mM,showing that lower percentages of DHT remaining are correlated withincreased nitric oxide donor (e.g., GSNO) concentrations. FIG. 74 is aplot of percentage of DHT remaining as a function of NO-donorconcentration (mM) for dark conditions and 420 nm light exposureconditions for NO-donor concentrations of 0 and 1 mM. Inhibition wasstill observed in the dark because the nitric oxide donor releasesnitric oxide under the conditions of the assay. FIG. 74 shows that lightdoes not have a detrimental effect on NO-induced inhibition. Asdemonstrated previously herein, modulated light therapy releases nitricoxide, which can then inhibit 5α-reductase and thereby provide atherapeutic benefit in terms of reduced (or reversed) hair loss forsuffers of androgenic alopecia and/or similar conditions.

Phototherapy has been shown to be effective in treating variousconditions, including alopecia, acne, seasonal affective disorder,psoriasis, excess bilirubin, atopic dermatitis, and a broad range ofaesthetic indications. While there may not be a single universallyaccepted mechanism for the biological activity of light, there may bemultiple biological mechanisms that are relevant depending on theintensity and wavelength of the therapeutic light. UV light may beconfigured to provide ultraviolet germicidal irradiation fordisinfecting of surfaces, food, air, and water. In such applications,the peak wavelength of light used may be in one or more wavelengthranges of the ultraviolet spectrum, for example 260 to 270 nm, which isunderstood to break bonds in DNA of microorganisms, thereby damaginggenetic material with fatal effect. While UV light is highly effectiveagainst microorganisms, it is non-selective and is known to also causedamage to human cells. In this regard, UV light provides someundesirable attributes that render it not universally suitable for allphototherapy applications. For visible light, such as in a range from400 nm to 700 nm, phototherapy has been suggested to provide therapeuticbenefits which include increasing circulation (e.g., by increasingformation of new capillaries); stimulating the production of collagen;stimulating the release of adenosine triphosphate (ATP); enhancingporphyrin production; reducing excitability of nervous system tissues;modulating fibroblast activity; increasing phagocytosis; inducingthermal effects; stimulating tissue granulation and connective tissueprojections; reducing inflammation; and stimulating acetylcholinerelease.

As previously described, phototherapy has also been suggested tostimulate cells to generate nitric oxide. Various biological functionsattributed to nitric oxide include roles as signaling messenger,cytotoxin, antiapoptotic agent, antioxidant, and regulator ofmicrocirculation. Nitric oxide is recognized to relax vascular smoothmuscles, dilate blood vessels, inhibit aggregation of platelets, andmodulate T cell-mediated immune response. Nitric oxide is produced bymultiple cell types in tissue and may be formed by the conversion of theamino acid L-arginine to L-citrulline and nitric oxide, mediated by theenzymatic action of nitric oxide synthases (NOSs).

Certain aspects of the present disclosure relate to phototherapeuticdelivery of light to mammalian tissue, including use of light having asingle peak wavelength and a single radiant flux or light havingmultiple peak wavelengths and/or multiple radiant fluxes to inhibit theprogression of a viral disease and/or to eradicate a viral infection.

In mammals, three distinct genes encode NOS isozymes: neuronal (nNOS orNOS-I), cytokine-inducible (iNOS or NOS-II), and endothelial (eNOS orNOS-III). iNOS and nNOS are soluble and found predominantly in thecytosol, while eNOS is membrane associated. Many cells in mammalssynthesize iNOS in response to inflammatory conditions. Systems andmethods for phototherapeutic modulation of nitric oxide has beendescribed in US Patent Application Publication No. 2017/0028216, whichis hereby incorporated by reference herein in its entirety.

In illustrative embodiments, provided are methods and exemplary devicesfor inactivating viruses in contact with tissue and/or treating aviral-infected tissue. In certain aspects, methods and correspondingdevices may include irradiating target tissue with a therapeutic dose(J/cm²) from a light source for a period of time, and repeating theirradiating step for a number (N) of iterations to constitute aduration, wherein N is an integer greater than 1. Irradiances of light(mW/cm²) have been proposed at specific wavelengths of visible light fora threshold time over a given duration to yield therapeutic dosages(J/cm²) which are effective for inactivating virus or treating viralinfections while maintaining the viability of epithelial tissues. Thesetreatments can be tailored to the particular tissue being treated, aswell as to the various fluids in the media, such as blood, sputum,saliva, cervical fluid, and mucous. The total dosage (J/cm²) to treat aninfection can be spread out over multiple administrations, with eachdose applied over seconds or minutes, and with multiple doses over daysor weeks, at individual doses that treat the infection while minimizingdamage to the particular tissue. Exemplary and nonlimiting RNA and DNAviruses that may be treated according to the principles of the presentdisclosure are summarized below.

There are currently 5 recognized orders and 47 families of RNA viruses,and there are also many unassigned species and genera. Related to butdistinct from the RNA viruses are the viroids and the RNA satelliteviruses.

There are several main taxa: levivirus and related viruses,picornaviruses, alphaviruses, flaviviruses, dsRNA viruses, and the -vestrand viruses (Wolf et al., “Origins and Evolution of the Global RNAVirome,” mBio, 9(6) (November 2018)).

Positive strand RNA viruses are the single largest group of RNA viruses,with 30 families. Of these, there are three recognized groups. Thepicorna group (Picornavirata) includes bymoviruses, comoviruses,nepoviruses, nodaviruses, picornaviruses, potyviruses, obemoviruses anda subset of luteoviruses (beet western yellows virus and potato leafrollvirus). The flavi-like group (Flavivirata) includes carmoviruses,dianthoviruses, flaviviruses, pestiviruses, statoviruses, tombusviruses,single-stranded RNA bacteriophages, hepatitis C virus and a subset ofluteoviruses (barley yellow dwarf virus). The alpha-like group(Rubivirata) includes alphaviruses, carlaviruses, furoviruses,hordeiviruses, potexviruses, rubiviruses, tobraviruses, tricornaviruses,tymoviruses, apple chlorotic leaf spot virus, beet yellows virus andhepatitis E virus.

A division of the alpha-like (Sindbis-like) supergroup has beenproposed, with two proposed groups. The ‘altovirus’ group includesalphaviruses, furoviruses, hepatitis E virus, hordeiviruses,tobamoviruses, tobraviruses, tricornaviruses and rubiviruses, and the‘typovirus’ group includes apple chlorotic leaf spot virus,carlaviruses, potexviruses and tymoviruses.

There are five groups of positive-stranded RNA viruses containing four,three, three, three, and one order(s), respectively. These fourteenorders contain 31 virus families (including 17 families of plantviruses) and 48 genera (including 30 genera of plant viruses).Alphaviruses and flaviviruses can be separated into two families, theTogaviridae and Flaviridae.

This analysis also suggests that the dsRNA viruses are not closelyrelated to each other but instead belong to four additional classes,Birnaviridae, Cystoviridae, Partitiviridae, and Reoviridae, and oneadditional order (Totiviridae) of one of the classes of positive ssRNAviruses in the same subphylum as the positive-strand RNA viruses.

There are two large clades: One includes the families Caliciviridae,Flaviviridae, and Picornaviridae and a second that includes the familiesAlphatetraviridae, Birnaviridae, Cystoviridae, Nodaviridae, andPermutotretraviridae. Satellite viruses include Albetovirus, Aumaivirus,Papanivirus, Virtovirus, and Sarthroviridae, which includes the genusMacronovirus.

Double-stranded RNA viruses (dsRNA viruses) include twelve families anda number of unassigned genera and species recognized in this group. Thefamilies include Amalgaviridae, Birnaviridae, Chrysoviridae,Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae,Partitiviridae, Picobirnaviridae, Reoviridae, which includes Rotavirus,Totiviridae, Quadriviridae. Botybirnavirus is one genus, and unassignedspecies include Botrytis porri RNA virus 1, Circulifer tenellus virus 1,Colletotrichum camelliae filamentous virus 1, Cucurbit yellowsassociated virus, Sclerotinia sclerotiorum debilitation-associatedvirus, and Spissistilus festinus virus 1.

Positive-sense ssRNA viruses (Positive-sense single-stranded RNAviruses) include three orders and 34 families, as well as a number ofunclassified species and genera. The order Nidovirales includes thefamilies Arteriviridae, Coronaviridae, which includes Coronaviruses,such as SARS-CoV and SARS-CoV-2, Mesoniviridae and Roniviridae. Theorder Picornavirales includes families Dicistroviridae, Iflaviridae,Marnaviridae, Picornaviridae, which includes Poliovirus, Rhinovirus (acommon cold virus), and Hepatitis A virus, Secoviridae, which includesthe subfamily Comovirinae, as well as the genus Bacillariornavirus andthe species Kelp fly virus. The order Tymovirales includes the familiesAlphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, and Tymoviridae.A number of families are not assigned to any of these orders, and theseinclude Alphatetraviridae, Alvernaviridae, Astroviridae, Barnaviridae,Benyviridae, Botourmiaviridae, Bromoviridae, Caliciviridae, whichincludes the Norwalk virus (i.e., norovirus), Carmotetraviridae,Closteroviridae, Flaviviridae, which includes Yellow fever virus, WestNile virus, Hepatitis C virus, Dengue fever virus, and Zika virus,Fusariviridae, Hepeviridae, Hypoviridae, Leviviridae, Luteoviridae,which includes Barley yellow dwarf virus, Polycipiviridae, Narnaviridae,Nodaviridae, Permutotetraviridae, Potyviridae, Sarthroviridae,Statovirus, Togaviridae, which includes Rubella virus, Ross River virus,Sindbis virus, and Chikungunya virus, Tombusviridae, and Virgaviridae.Unassigned genuses include Blunervirus, Cilevirus, Higrevirus,Idaeovirus, Negevirus, Ourmiavirus, Polemovirus, Sinaivirus, andSobemovirus. Unassigned species include Acyrthosiphon pisum virus,Bastrovirus, Blackford virus, Blueberry necrotic ring blotch virus,Cadicistrovirus, Chara australis virus, Extra small virus, Goji berrychlorosis virus, Harmonia axyridis virus 1, Hepelivirus, Jingmen tickvirus, Le Blanc virus, Nedicistrovirus, Nesidiocoris tenuis virus 1,Niflavirus, Nylanderia fulva virus 1, Orsay virus, Osedax japonicus RNAvirus 1, Picalivirus, Planarian secretory cell nidovirus, Plasmoparahalstedii virus, Rosellinia necatrix fusarivirus 1, Santeuil virus.Secalivirus, Solenopsis invicta virus 3, and Wuhan large pig roundwormvirus.

Satellite viruses include the family Sarthroviridae and the genusesAlbetovirus, Aumaivirus, Papanivirus, Virtovirus, and the Chronic beeparalysis virus. Six classes, seven orders and twenty four families arecurrently recognized in this group. A number of unassigned species andgenera are yet to be classified.

Negative-sense ssRNA viruses (Negative-sense single-stranded RNAviruses) are, with the exception of the Hepatitis D virus, within asingle phylum, Negarnaviricota, with two subphyla, Haploviricotina andPolyploviricotina, with four classes, Chunqiuviricetes, Milneviricetes,Monjiviricetes and Yunchangviricetes. The subphylum Polyploviricotinahas two classes, Ellioviricetes and Insthoviricetes.

There are also a number of unassigned species and genera. The PhylumNegarnaviricota includes Subphylum Haploviricotina, ClassChunqiuviricetes, Order Muvirales, Family Qinviridae. The ClassMilneviricetes includes Order Serpentovirales and Family Aspiviridae.The Class Monjiviricetes includes Order Jingchuvirales and FamilyChuviridae. The order Mononegavirales includes families Bornaviridae,which includes the Borna disease virus, Filoviridae, which includes theEbola virus and the Marburg virus, Mymonaviridae, Nyamiviridae,Paramyxoviridae, which includes Measles, Mumps, Nipah, Hendra, and NDV,Pneumoviridae, which RSV and Metapneumovirus, Rhabdoviridae, whichRabies, and Sunviridae, as well as genuses Anphevirus, Arlivirus,Chengtivirus, Crustavirus, and Wastrivirus. Class Yunchangviricetesincludes order Goujianvirales and family Yueviridae. SubphylumPolyploviricotina includes class Ellioviricetes, order Bunyavirales, andthe families Arenaviridae, which includes Lassa virus, Cruliviridae,Feraviridae, Fimoviridae, Hantaviridae, Jonviridae, Nairoviridae,Peribunyaviridae, Phasmaviridae, Phenuiviridae, Tospoviridae, as well asgenus Tilapineviridae.

Class Insthoviricetes includes order Articulavirales and familyAmnoonviridae, which includes the Taastrup virus, and familyOrthomyxoviridae, which includes Influenza viruses.

The genus Deltavirus includes the Hepatitis D virus.

Specific viruses include those associated with infection of mucosalsurfaces of the respiratory tract, including Betacoronavirus (SARS-CoV-2and MERS-CoV), rhinoviruses, influenza virus (including influenza A andB, parainfluenza). Generally, orthomyxoviruses and paramyxoviruses canbe treated.

A DNA virus is a virus that has DNA as its genetic material andreplicates using a DNA-dependent DNA polymerase. The nucleic acid isusually double-stranded DNA (dsDNA) but may also be single-stranded DNA(ssDNA). DNA viruses belong to either Group I or Group II of theBaltimore classification system for viruses. Single-stranded DNA isusually expanded to double-stranded in infected cells. Although GroupVII viruses such as hepatitis B contain a DNA genome, they are notconsidered DNA viruses according to the Baltimore classification, butrather reverse transcribing viruses because they replicate through anRNA intermediate. Notable diseases like smallpox, herpes, and thechickenpox are caused by such DNA viruses.

Some DNA viruses have circular genomes (Baculoviridae, Papovaviridae andPolydnaviridae) while others have linear genomes (Adenoviridae,Herpesviridae and some phages). Some families have circularly permutedlinear genomes (phage T4 and some Iridoviridae). Others have lineargenomes with covalently closed ends (Poxviridae and Phycodnaviridae).

Fifteen DNS virus families are enveloped, including all three familiesin the order Herpesvirales and the following families: Ascoviridae,Ampullaviridae, Asfarviridae, Baculoviridae, Fuselloviridae,Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae,Lipothrixviridae, Nimaviridae and Poxviridae.

Of these, species of the order Herpesvirales, which includes the familesAlloherpesviridae, Herpesviridae, which includes human herpesviruses andthe Varicella Zoster, and the families Adenoviridae, which includesviruses which cause human adenovirus infection, and Malacoherpesviridae,infect vertebrates.

Asfarviridae, which includes African swine fever virus, Iridoviridae,Papillomaviridae, Polyomaviridae, which includes Simian virus 40, JCvirus, and BK virus, and Poxviridae, which includes Cowpox virus andsmallpox, infect vertebrates. Anelloviridae and Circoviridae also infectanimals (mammals and birds respectively).

The family Smacoviridae includes a number of single-stranded DNA virusesisolated from the feces of various mammals, and there are 43 species inthis family, which includes six genera, namely, Bovismacovirus,Cosmacovirus, Dragsmacovirus, Drosmacovirus, Huchismacovirus andPorprismacovirus. Circo-like virus Brazil hs1 and hs2 have also beenisolated from human feces. An unrelated group of ssDNA viruses includesthe species bovine stool associated circular virus and chimpanzee stoolassociated circular virus.

Animal viruses include parvovirus-like viruses, which have linearsingle-stranded DNA genomes, but unlike the parvoviruses, the genome isbipartate. This group includes Hepatopancreatic parvo-like virus andLymphoidal parvo-like virus. Parvoviruses have frequently invaded thegerm lines of diverse animal species including mammals.

The human respiratory-associated PSCV-5-like virus has been isolatedfrom the respiratory tract. The PSCV-50-like virus may also be subjectto phototherapy principles of the present disclosure.

According to certain embodiments, provided herein are methods oftreating and/or preventing a viral infection. A method of treatingand/or preventing a viral infection may comprise administering light tothe skin of a subject, thereby treating and/or preventing the viralinfection in the subject. In some embodiments, aspects of the presentinvention may provide suppression and/or inhibition of viral replicationand/or enhancement of local immune responses of a subject.

According to certain embodiments of the present disclosure, providedherein are methods and devices of treating and/or preventingvirus-related cutaneous conditions. A method of treating and/orpreventing a virus-related cutaneous condition may compriseadministering light to the skin of a subject, thereby treating and/orpreventing the virus-related cutaneous condition in the subject.Virus-related cutaneous conditions that may be treated and/or preventedinclude, but are not limited to, cutaneous conditions associated withbowenoid papulosis, buffalopox, butcher's wart, condylomata acuminate,cowpox, cytomegalovirus, disseminated herpes zoster, eczema herpeticum(Kaposi's varicelliform eruption), eczema vaccinatum, epidermodysplasiaverruciformis, erythema infectiosum (fifth disease, slapped cheekdisease), farmyard pox, generalized vaccinia, genital herpes (herpesgenitalis, herpes progenitalis), Buschke-Löwenstein tumor,hand-foot-and-mouth disease (Coxsackie), Heck's disease (focalepithelial hyperplasia), herpangina, herpes gladiatorum (scrum pox),herpes simplex, herpetic keratoconjunctivitis, herpetic sycosis,herpetic whitlow, human monkeypox, human T-lymphotropic virus 1infection, human tanapox, intrauterine herpes simplex, Kaposi sarcoma,Lipschtltz ulcer (ulcus vulvae acutum), Milker's nodule, molluscumcontagiosum, neonatal herpes simplex, ophthalmic zoster, orf (contagiouspustular dermatosis, ecthyma contagiosum, infectious labial dermatitis,sheep pox), oral florid papillomatosis, oral hairy leukoplakia (EBV),orolabial herpes (herpes labialis), progressive vaccinia (vacciniagangrenosum, vaccinia necrosum), pseudocowpox, recurrent respiratorypapillomatosis (laryngeal papillomatosis), sealpox, varicella(chickenpox), variola major (smallpox), verruca plana (flat warts),verruca plantaris (plantar wart), verruca vulgaris (wart), verrucaepalmares et plantares, and/or zoster (herpes zoster, shingles). In someembodiments, the viral infection may be caused by a papillomavirus, suchas a human papillomavirus. The human papillomavirus (HPV) may be HPVtype 1, 2, 3, 4, 6, 10, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58,and/or 59.

In certain embodiments, methods and/or devices for treating and/orpreventing a virus-related gastrointestinal (GI) condition may compriseadministering light via colorectal administration via a probe insertedinto the body cavity of a subject, thereby treating and/or preventingthe virus-related colorectal or intestinal condition in the subject.Viruses in the GI tract include rotavirus, picornavirus, andcoronavirus. In one embodiment the condition is caused by EnterovirusD68 known to cause severe illness in children and acute flacid myelitis.

In other embodiments, methods and/or devices for treating and/orpreventing a virus-related central nervous system (CNS) infection maycomprise administering light transcranially, through the nose of apatient, or upon implantation of a light source into the tissue of asubject, thereby treating and/or preventing the virus-related CNScondition in the subject. CNS infections are frequently caused byviruses, such as the enteroviruses, which cause the majority of cases ofaseptic meningitis and meningoencephalitis as well as other neurotropicviruses including but not limited to human cytomegalovirus, herpessimplex viruses, varicella-zoster virus, and the emerging viruses WestNile virus, Murray Valley encephalitis virus, henipaviruses, Japaneseencephalitis virus, chikungunya virus, Ebola virus, and rabies virus.

In specific embodiments, intranasal administration to the nasal mucosacan be used as a method of treating and/or preventing a virus-relatedinfection. Data from animal studies and human cases have demonstratedthat the olfactory and/or trigeminal nerve pathway represents a majorroute of CNS entry for several groups of viruses. It is known thatherpes simplex virus type 1, bovine herpesvirus 5, and equineherpesvirus 9 spread from the nasal mucosa to the CNS via the olfactorynerves in animal models of infection. Orthomyxoviridae, includinginfluenza virus, is also spread from the nasal cavity to the olfactorybulb and the rest of the CNS. Paramyxoviruses, including Nipah virus,Hendra virus, and parainfluenza virus, may enter the CNS directly fromthe nasal mucosa.

According to other embodiments of the present disclosure, methods and/ordevices for treating and/or preventing a virus-related bloodstreaminfection may comprise transdermal administration of light tosuperficial vasculature, administering light to blood passed through anextra-corporeal loop, shining light on a blood product derived from thepatient for use on other patients, and other methods for illumination ofbiological fluids of a subject, thereby treating and/or preventing thevirus-related blood stream infection in the subject. In someembodiments, the biological fluid is treated to help treat or preventviremia, which is when viruses are present in the blood at abnormallevels. Viremia can be classified into primary viremia, the spread ofthe virus into the blood from the initial site of infection or secondaryviremia, the spread of the virus to other organs that come into contactwith the blood where the virus replicates and then enters thebloodstream once more. In some methods, the viremia may be active. Inother embodiments, the viremia may be passive. In some embodiments, theviremia is caused by West Nile virus, dengue, rubella, measles,cytomegalovirus, Epstein-Barr virus, HIV, hepatitis B virus, poliovirus,yellow fever virus, or varicella-zoster virus.

In other embodiments, the light is applied external to the body to thejoints including those in the feet and hands, as well as the ankles,elbows, knees, and shoulders as a method of treating and/or preventing ajoint arthritis related to side effects caused by autoimmune reactionsto viruses including but not limited to chikungunya and ross rivervirus.

The terms phototherapy and phototherapeutic relate to the therapeuticuse of light. As used herein, phototherapy is used to treat or preventmicrobial infections, including viral infections of the body includingmucosal epithelial tissues in the vaginal cavity, anal canal, oralcavity, the auditory canal, the upper respiratory tract and esophagus.

The mechanisms by which the wavelengths of light are effective can vary,depending on the wavelength that is administered. Biological effects,including antimicrobial effects, can be provided over a wide range ofwavelengths, including UV ranges, visible light ranges, and infraredranges. The effects vary depending on the mechanism by which the lightis antimicrobial, and the wavelengths that bring about these mechanisms.Phot

A handful of photoacceptors for blue light have been identified innon-pigmented cells, including cytochrome c oxidase, flavins,porphyrins, opsins, and nitrosated proteins. Light absorption byphotoreceptors can lead to release of reactive oxygen species (ROS)and/or nitric oxide (NO) that may function to inactivate viruses in acell-free or cell-associated environment. Reactive oxygen species and/orbioactive NO may elicit activation of transcription factors involved inimmune signaling, such as nuclear factor kappa-light-chain-enhancer ofactivated B cells (NF-KB) and mitogen activated protein kinase (MAPK)signaling. NFKB and MAPK pathways can lead to transcriptional activationof innate and inflammatory immune response molecules that may interferewith viral replication. Nitric oxide may also mediate inactivation ofcell-associated virus through S-nitrosylation of cysteine residues inthe active site of viral encoded enzymatic proteins. Reactive oxygenspecies and/or NO may also function to inactivate cell-free virions.Photosensitizers present in cell media may facilitate generation of ROSand/or NO that directly impact virion proteins and/or viral RNA toprevent infection and replication. Evidence demonstrating that SARS-CoVcan be inactivated by exogenous addition of NO donor moleculessubstantiate the potential for SARS-CoV-2 inactivation by nitric oxide.

In some embodiments, the wavelengths of light activate immune cells ofthe innate and/or adaptive immune responses, including macrophages.

When administering light to arrive at a suitable total dose (J/cm²), itcan be important to provide the therapeutic dosage of light at asuitable combination of a wavelength, and irradiance (W/cm²) to thetarget tissue, and exposure time, and multiple exposures, at theseconditions to yield total dose in J/cm².

The wavelength should be safe to the tissue being irradiated, and theirradiance should be safe to the tissue as well, ideally not heating thetissue to a temperature that is unsafe, and the cumulative exposure timeshould be matched with the desired clinical application. In someembodiments, the device used to administer the light can include a meansfor controlling the amount of light that is administered, such as atimer, actuator, dosimeter, and the like, such that the light does notexceed safe limits.

For example, light is ideally administered at a dosage that is safe andat a dosage that is effective at killing viruses or other microbes. Inthis regard, aspects of the present disclosure provide a ratio of theIC₂₅ (the concentration or dose required to reduce living tissueviability by 25% when compared to control-treated tissues) to the EC₅₀(dose required to kill 50% of the virus or other microbe for thespecific tissue being treated as quantified at a cellular level) isgreater than or equal to 2. As disclosed herein, the IC₂₅/EC₅₀ ratio orfraction may be referred to as a light therapeutic index (LTI) thatquantifies safe and effective light dosages. In another context, one canconsider, in an in vitro setting, the ratio of the CC₅₀ (concentrationof a therapeutic to reduce cell viability by 50%) to the EC₅₀ fortreated cells (i.e., the Selectivity Index, or “SI”). This ratio willvary depending on the type of cells or tissue that are exposed, forexample, with some cells having differential tolerance to oxidativedamage than other cells.

Phototherapeutic light can induce oxidative and nitrosative stresseswithin cells or the tissues comprised therefrom. Referring now to FIG.75, shown is a diagrammatic illustration of proposed mechanisms ofaction whereby phototherapy leads to inhibited cell proliferation,altered terminal differentiation, and apoptosis. Illustratively, theReactive Oxygen Species (ROS) or Reactive Oxygen Pathway causesoxidative stress through either Type 1 or Type 2 Photoreactions. Type 1reactions involve the excitation of an endogenous photosensitizer (mostlikely an endogenous porphyrin). This excited photosensitizer reactswith another cellular component to form a free radical directly (e.g.superoxide and hydroxyl groups). Type 2 reactions involve the excitationof an endogenous photosensitizer (again, most likely a porphyrin) withoxygen to form singlet oxygen (1O2). It is understood that phototherapycauses increased ROS levels which can create oxidative stress in thesubject tissue. Virus-infected tissues are known to already be underconsiderable oxidative stress due to their viral infections. Theincrease in oxidative stress through phototherapy thus exacerbates theoxidative environment and depending on phototherapy dosage, can inhibitcell proliferation, alter terminal differentiation, and cause apoptosis.

Referring back to FIG. 75, phototherapy can also induce a nitric oxidepathway which can create nitrosative stress. The nitrosative stress isinduced by the increase in the enzymatic generation of nitric oxide andthe photodissociation of endogenous nitric oxide. Both enhancing theenzymatic generation of nitric oxide and the photodissociation of nitricoxide from the Endogenous Stores (ES), result in increased NO levels. Itis understood that this nitrosative stress likewise can inhibit cellproliferation, alter terminal differentiation, and cause apoptosis.

As disclosed herein, high doses of blue light are shown to increase theexpression of nitric oxide synthase enzymes. Furthermore, thephotodissociation of NO from endogenous stores is well known. Therelease of NO from nitrosated/nitrosylated proteins is possible througha wavelength dependent process as described in U.S. Patent ApplicationPublication No. 2017/0028215, which is incorporated by reference hereinin its entirety for disclosure associated with phototherapy.

The photoinitiated release of endogenous stores of nitric oxideeffectively regenerates “gaseous” (or unbound) nitric oxide that may beautooxidized into nitrosative intermediates and bound covalently in thebody in a “bound” state. By stimulating release of nitric oxide fromendogenous stores, nitric oxide may be maintained in a gaseous state foran extended duration and/or a spatial zone of nitric oxide release maybe expanded.

Another aspect of the present disclosure is that one or more viruses canbe inactivated pre-infection and one or more viral infections can beinhibited and/or eradicated by light-induced nitrosative or oxidativestress.

An illumination device for the treatment of pathogen infected tissuesand/or for the inducing one or more biological effects, may take anyform suitable for delivering light to the infected tissue. The devicewill contain a light source capable of emitting a suitable light profilethat can provide one or more direct or indirect biological effects. Alight profile can be represented with a graph of emission intensityversus wavelength of light for any particular light source. Disclosedherein are light sources with light profiles in the visible spectrum,for example with light emissions with peak wavelengths primarily in arange from 400 nm to 700 nm. Depending on the target application, lightprofiles may also include infrared or near-infrared peak wavelengths ator above 700 nm including up to 900 nm, or ultraviolet peak wavelengthsat or below 400 nm including as low as 200 nm. In certain embodiments,light emissions may have a single peak wavelength in a range from 200 nmto 900 nm, or in a range from 400 nm to 490 nm, or in a range from 400nm to 450 nm, or in a range from 400 nm to 435 nm, or in a range from400 nm to 420 nm, or in a range from 410 nm to 440 nm, or in a rangefrom 420 nm to 440 nm, or in a range from 450 nm to 490 nm, or in arange from 500 nm to 900 nm, or in a range from 490 nm to 570 nm, or ina range from 510 nm to 550 nm, or in a range from 520 nm to 540 nm, orin a range from 525 nm to 535 nm, or in a range from 528 nm to 532 nm,or in a range from 320 nm to 400 nm, or in a range from 350 nm to 395nm, or in a range from 280 nm to 320 nm, or in a range from 320 nm to350 nm, or in a range from 200 nm to 280 nm, or in a range from 260 nmto 270 nm, or in a range from 240 nm to 250 nm, or in a range from 200nm to 225 nm. In further embodiments, light emissions may includemultiple peak wavelengths selected from any of the above listed ranges,depending on the target application and desired biological effects.Depending on the target application, full width half maximum (FWHM)values for any of the above-described peak wavelength ranges may be lessthan or equal to 100 nm, or less than or equal to 90 nm, or less than orequal to 40 nm, or less than or equal to 20 nm. In certain aspects,lower FWHM values are typically associated with single emission colorLEDs in any of the above-described wavelength bands. Larger FWHM values(e.g., from 40 nm to 100 nm) may be associated with phosphor-convertedLEDs where spectral bandwidths are a combination of LED emissions andphosphor-converted emissions. Exemplary phosphor-converted LEDs that maybe applicable to the present disclosure are phosphor-converted amberLEDs having peak wavelengths in a range from 585 nm to 600 nm and FWHMvalues in a range from 70 nm to 100 nm, and phosphor-converted mintand/or lime LEDs having peak wavelengths in a range from 520 nm to 560nm. Additional embodiments of the present disclosure may also beapplicable to broad spectrum white LEDs that may include an LED with apeak wavelength in a range from 400 nm to 470 nm, and one or morephosphors to provide the broad emission spectrum. In such embodiments, abroad spectrum LED may provide certain wavelengths that induce one ormore biological effects while also providing broad spectrum emissions tothe target area for illumination. In this regard, light impingement ontissue for single and/or multiple microorganism and/or multiplepathogenic biological effects may be provided with light of a singlepeak wavelength or a combination of light with more than one peakwavelength.

Doses of light to induce one or more biological effects may beadministered with one or more light characteristics, including peakwavelengths as described above, radiant flux, and irradiance to targettissues. Irradiances to target tissues may be provided in a range from0.1 mW/cm² to 200 mW/cm², or in a range from 5 mW/cm² to 200 mW/cm², orin a range from 5 mW/cm² to 100 mW/cm², or in a range from 5 mW/cm² to60 mW/cm², or in a range from 60 mW/cm² to 100 mW/cm², or in a rangefrom 100 mW/cm² to 200 mW/cm². Such irradiance ranges may beadministered in one or more of continuous wave and pulsedconfigurations, including LED-based photonic devices that are configuredwith suitable power (radiant flux) to irradiate a target tissue with anyof the above-described ranges. A light source for providing suchirradiance ranges may be configured to provide radiant flux values fromthe light source of at least 5 mW, or at least 10 mW, or at least 15 mW,or at least 20 mW, or at least 30 mW, or at least 40 mW, or at least 50mW, or at least 100 mW, or at least 200 mW, or in a range of from 5 mWto 200 mW, or at least 500 mW, or at least 2500 mW, or at least 5000 mw,or in a range of from 5 mW to 100 mW, or in a range of from 5 mW to 60mW, or in a range of from 5 mW to 30 mW, or in a range of from 5 mW to20 mW, or in a range of from 5 mW to 10 mW, or in a range of from 10 mWto 60 mW, or in a range of from 20 mW to 60 mW, or in a range of from 30mW to 60 mW, or in a range of from 40 mW to 60 mW, or in a range of from60 mW to 100 mW, or in a range of from 100 mW to 200 mW, or in a rangeof from 200 mW to 500 mW, or in a range of from 5 mW to 5000 mW, or in arange of from 5 mW to 2500 mW, or in another range specified herein.Depending on the configuration of one or more of the light source, thecorresponding illumination device, and the distance away from a targettissue, the radiant flux value for the light source may be higher thanthe irradiance value at the tissue. In certain embodiments, the radiantflux value may be configured with a value that is greater than theirradiance value to the tissue. For example, the radiant flux may be ina range from 5 to 20 times greater than the irradiance, or in a rangefrom 5 to 15 times greater than the irradiance, among other ranges anddepending on the embodiments.

While certain peak wavelengths for certain target tissue types may beadministered with irradiances up to 1 W/cm²without causing significanttissue damage, safety considerations for other peak wavelengths andcorresponding tissue types may require lower irradiances, particularlyin continuous wave applications. In certain embodiments, pulsedirradiances of light may be administered, thereby allowing safeapplication of significantly higher irradiances. Pulsed irradiances maybe characterized as average irradiances that fall within safe ranges,thereby providing no or minimal damage to the applied tissue. In certainembodiments, irradiances in a range from 0.1 W/cm² to 10 W/cm² may besafely pulsed to target tissue.

Administered doses of light, or light doses, may be referred to astherapeutic doses of light in certain aspects. Doses of light mayinclude various suitable combinations of the peak wavelength, theirradiance to the target tissue, and the exposure time period.Particular doses of light are disclosed that are tailored to providesafe and effective light for inducing one or more biological effects forvarious types of pathogens and corresponding tissue types. In certainaspects, the dose of light may be administered within a single timeperiod in a continuous or a pulsed manner. In further aspects, a dose oflight may be repeatably administered over a number of times to provide acumulative or total dose over a cumulative time period. By way ofexample, a single dose of light as disclosed herein may be provided overa single time period, such as in a range from 10 microseconds to no morethan an hour, or in a range from 10 seconds to no more than an hour,while the single dose may be repeated at least twice to provide acumulative dose over a cumulative time period, such as a 24-hour timeperiod. In certain embodiments, doses of light are described that may beprovided in a range from 0.5 joules per square centimeter (J/cm²) to 100J/cm², or in a range from 0.5 J/cm² to 50 J/cm², or in a range from 2J/cm² to 80 J/cm², or in a range from 5 J/cm² to 50 J/cm², whilecorresponding cumulative doses may be provided in a range from 1 J/cm²to 1000 J/cm², or in a range from 1 J/cm² to 500 J/cm², or in a rangefrom 1 J/cm² to 200 J/cm², or in a range from 1 J/cm² to 100 J/cm², orin a range from 4 J/cm² to 160 J/cm², or in a range from 10 J/cm² to 100J/cm², among other discloses ranges. In a specific example, a singledose may be administered in a range from 10 J/cm² to 20 J/cm², and thesingle dose may be repeated twice a day for four consecutive days toprovide a cumulative dose in a range from 80 J/cm² to 160 J/cm². Inanother specific example, a single dose may be administered at about 30J/cm², and the single dose may be repeated twice a day for sevenconsecutive days to provide a cumulative dose of 420 J/cm².

In still further aspects, light for inducing one or more biologicaleffects may include administering different doses of light to a targettissue to induce one or more biological effects for different targetpathogens. As disclosed herein, a biological effect may include alteringa concentration of one or more pathogens within the body and alteringgrowth of the one or more pathogens within the body. The biologicaleffect may include at least one of inactivating the first pathogen in acell-free environment, inhibiting replication of the first pathogen in acell-associated environment, upregulating a local immune response in themammalian tissue, stimulating enzymatic generation of nitric oxide toincrease endogenous stores of nitric oxide in the mammalian tissue,releasing nitric oxide from endogenous stores of nitric oxide in themammalian tissue, and inducing an anti-inflammatory effect in themammalian tissue. As further disclosed herein, a pathogen may include avirus, a bacteria, and a fungus, or any other types of microorganismsthat can cause infections. Notably, light doses as disclosed herein mayprovide non-systemic and durable effects to targeted tissues. Light canbe applied locally and without off-target tissue effects or overallsystemic effects associated with conventional drug therapies which canspread throughout the body. In this regard, phototherapy may induce abiological effect and/or response in a target tissue without triggeringthe same or other biological responses in other parts of the body.Phototherapy as described herein may be administered with safe andeffective doses that are durable. For example, a dose may be applied forminutes at time, one to a few times a day, and the beneficial effect ofthe phototherapy may continue in between treatments.

Light sources may include one or more of LEDs, OLEDs, lasers and otherlamps according to aspects of the present disclosure. Lasers may be usedfor irradiation in combination with optical fibers or other deliverymechanisms. A disadvantage of using a laser is that it may requiresophisticated equipment operated by highly skilled professionals toensure proper laser radiation protection, thereby increasing costs andreducing accessibility. LEDs are solid state electronic devices capableof emitting light when electrically activated. LEDs may be configuredacross many different targeted emission spectrum bands with highefficiency and relatively low costs. In this regard, LEDs arecomparatively simpler devices that operate over much wider ranges ofcurrent and temperature, thereby providing an effective alternative toexpensive laser systems. Accordingly, LEDs may be used as light sourcesin photonic devices for phototherapy applications. Light from an LED isadministered using a device capable of delivering the requisite power toa targeted treatment area or tissue. High power LED-based devices can beemployed to fulfill various spectral and power needs for a variety ofdifferent medical applications. LED-based photonic devices describedherein may be configured with suitable power to reach power densities ashigh as 100 mW/cm² or 200 mW/cm² in the desired wavelength range. An LEDarray in this device can be incorporated into an irradiation head, handpiece and or as an external unit. When incorporated into hand piece orirradiation head, risk of eye or other organs being exposed to harmfulradiation may be avoided.

According to aspects of the present disclosure, exemplary target tissuesand cells light treatments may include one or more of epithelial tissue,mucosal tissue, connective tissue, muscle tissue, cervical tissue,dermal tissue, mucosal epithelial tissues in the vaginal cavity, analcanal, oral cavity, the auditory canal, the upper respiratory tract andesophagus, keratinocytes, fibroblasts, blood, sputum, saliva, cervicalfluid, and mucous. Light treatments may also be applied to and/or withinorgans, to external body surfaces, and within any mammalian body and/orbody cavity, for example the oral cavity, esophageal cavity, throat, andvaginal cavity, among others.

In certain aspects, effective phototherapy-based treatment modalitiesare provided for precancerous and cancerous conditions of portio andcervical regions of a female anatomy, as well as tissues in the anus,throat and mouth of both sexes. By way of illustration, an illustrationof the female reproductive system is shown in FIG. 76. Certain oncogenichuman papilloma virus (HPV) can infect the cells in the cervix andportio region inducing dysplasias that can develop into cancerousconditions when left untreated. In the US alone, there are around half amillion patients surgically treated per year for colonization of theportio. This virus can also cause mouth, throat and anal cancers. Theregion of the cervix where the columnar epithelium has been replaced bythe new metaplastic squamous epithelium is referred to as thetransformation zone. Identifying the transformation zone is important asalmost all manifestations of cervical carcinogenesis occur in this zone.

Exemplary devices for delivering phototherapy within body cavities aredescribed below with regard to FIGS. 77A, 77B, 78A, and 78B. Whilevarious aspects of such exemplary devices are provided, it is understoodthat phototherapy and light treatments according to the principles ofthe present disclosure may be administered by many different types ofdevices beyond the examples provided below. Devices for administeringphototherapy and light treatments as described herein may embodyhand-held devices, disposable devices, and devices that are incorporatedor attached with larger medical equipment, among other device types.Additionally, such devices may be configured for partial or completeinsertion within one or more body cavities. The phototherapy devices asdescribed below for FIGS. 77A-78B may include any of the components asdescribed for FIG. 40, including the microcontroller, the battery, theboost circuits, the charging integrated circuit, the microUSB connector,the user input buttons, the temperature and/or proximity sensor aspreviously described.

FIG. 77A illustrates an internal view of an exemplary illuminationdevice 700 as described herein. As illustrated, the illumination device700 may include a structure, such as a wand, that can be at leastpartially inserted into one or more body cavities, includingintravaginally, intra-anally, orally, or within the nasal cavity. At oneend, the illumination device 700 may include a lens optic 702 that isarranged within an illumination head 704. The lens optic 702 may beconfigured to pass light toward a target tissue and in certainembodiments, the lens optic 702 may be provided in optical communicationwith a camera for viewing the target tissue. The illumination head 704may be coupled to a rotation yoke 706, thereby allowing the illuminationhead 704 to be adjustable to a number of tilt and/or rotation anglesrelative to the remainder of the illumination device 700. In certainembodiments, the illumination head 704 may be configured to tilt, angle,and/or rotate as much as 60 degrees from a lengthwise direction of theillumination device 700. The illumination device 700 may include aninflation bladder 708 that may be inflated for holding the illuminationdevice 700 in place after insertion into a body cavity. A light sourcehousing 710 may be provided at an opposite end of the illuminationdevice 700 to the illumination head 704. For LED embodiments, the lightsource housing 710 may include an LED board 712 that is populated withone or more LEDs that are configured to provide any of the wavelengthranges described above. In particular, the LED board 712 may beconfigured to provide a single target peak wavelength or a plurality ofdifferent target peak wavelengths, depending on the application. A lightguide housing 714 that includes at least one light guide is arrangedbetween the illumination head 704 and the light source housing 710 todirect light from the LEDs to the illumination head 704. The light guidemay include one or more of a waveguide and a fiber optic material. Asheath 716 may form a protective covering over the illumination head 704and the light guide housing 714 along areas of the illumination device700 that may be inserted within a body cavity. In certain embodiments,the sheath 716 may comprise silicone. In this regard, the LEDs (e.g., onthe LED board 712) may be positioned outside of a body cavity and lightfrom the LEDs may be delivered through the illumination head 704 thatresides within the body cavity. The LED board 712 may further comprisedriver circuitry for driving the LEDs. In certain embodiments, ascrew-on locking cap 718 may be provided between the light guide housing714 and the light source housing 710. The screw-on locking cap 718 maybe configured to allow the light guide housing 714 and illumination head704 to be removably attached to the light source housing 710. Theillumination device 700 may further include a set screw 720, aninsulation/wiring chamber 722, a flex cable 724 (e.g., braided polyvinylchloride (PVC)) and corresponding strain relief 726, electrical wiring728 (e.g., DC wiring), a video cable 730, and an air line 732 forinflating the inflation bladder 708. Circuitry for the camera, such as acamera printed circuit board 734, may be arranged within one or more ofthe light guide housing 714 and the light source housing 710.

FIG. 77B illustrates an external view of the illumination device 700 ofFIG. 77A. As illustrated, the inflation bladder 708 may be inflated andhold the illumination device 700 in place within a body cavity, forexample within the vaginal canal, the anus, the oral cavity, and/or thenasal cavity. In certain embodiments, grip ridges 736 may be formed inthe sheath 716 so that the sheath 716 may be rolled back from the lightsource housing 710, for ease of cleaning between uses. As describeabove, the light guide housing 714 and illumination head 704 may form adetachable shaft that can be removably attached to the light sourcehousing 710 by way of the locking cap 718. Dimensions of the detachableshaft may be determined based on the target body cavity. By way ofexample, for use in the vaginal canal, the detachable shaft may includea length L1 in a range from 75 mm to 160 mm, or in a range from 140 mmto 160 mm as measured by a length of the sheath 716 from the locking cap718 to an end of the illumination device 700 at the illumination head704. In certain embodiments, a combination of the inflation bladder 708and the illumination head 704 may encompass a sub-dimension orsub-length L2 of the length L1 of the detachable shaft that is in arange from 25 mm to 55 mm. A grip ring 738, such as a polycarbonatering, may be attached to the locking cap 718. The illumination device700 may further include an adjustment control 740, such as a button orslide switch, that allows a user to adjust an angle of the illuminationhead 704 during operation so that the direction of light emissionsand/or the direction of the camera are adjustable. The adjustmentcontrol 740 may further be lockable once a desired angle of theillumination head 704 is achieved.

FIG. 78A illustrates an internal view of an illumination device 742 thatmay be disposable according to principles of the present disclosure. Theillumination device 742 may include a light source 744 that is coveredby an optic 746. The light source 744 may include one or more single ormultiple-LED packages that are configured to provide a single targetpeak wavelength or a plurality of different target peak wavelengths,depending on the application. While not shown, the single ormultiple-LED packages may optionally include one or more lenses. Theoptic 746 may comprise silicone, e.g., a liquid silicone rubber and thelike, and the optic 746 may form any shape for directing emissions fromthe light source 744 in a desired direction and/or pattern. By way ofexample, the optic 746 in FIG. 78A forms an angled shape that maypreferentially direct emissions in a non-perpendicular direction from amounting plane of the light source 744. The light source 744 may be atleast partially encased in a protective covering 748 that together withthe optic 746 seals the light source 744 within the illumination device742. In certain embodiments, the protective covering 748 may compriseclear or light-transmissive material, such as silicone. In certainembodiments, the optic 746 is an integral single piece with theprotective covering 748. Stated differently, a portion of the protectivecovering 748 may form the optic 746 in such embodiments. A heat sink 750may be provided within the protective covering 748 and in thermalcommunication with the light source 744. The heat sink 750 may compriseany material with high thermal conductivity relative to other elementsof the illumination device 742. In certain embodiments, the light source744 may generate heat during operation and the heat sink 750 mayaccordingly form a heat pipe that provides a heat dissipation path awayfrom the light source 744. A thermal sensor 752 and correspondingcircuitry may also be provided within the protective covering 748 thatis configured to electrically deactivate the light source 744 if a safepredetermined operating temperate range is exceeded. A cable 754 mayalso be arranged to power and control the light source 744.Additionally, the cable 754 may be used to retrieve the illuminationdevice 742 from a body cavity after completion of phototherapy. In thisregard, the illumination device 742 may be configured to be fullyinserted within a body cavity, but for a portion of the cable 754. Incertain embodiments, the protective covering 748 may also encase one ormore portions of the cable 754. The illumination device 742 as describedherein may be configured as a disposable device with a form factor thatis suitable for single use and/or a limited number of uses. Dimensionsof the illumination device 742 may be determined based on the targetbody cavity. By way of example, in certain embodiments, a length L3 asmeasured from an end of the optic 746 to the cable 754 may be providedin a range from 20 mm to 100 mm, or in a range from 20 mm to 80 mm, orin a range from 30 mm to 70 mm, or in a range from 40 mm to 60 mm. Incertain embodiments, a height H or diameter of the illumination device742 may be provided in a range from 1 mm to 20 mm, or in a range from 1mm to 15 mm, or in a range from 5 mm to 15 mm.

FIG. 78B illustrates an internal view of another illumination device 756that may be disposable according to principles of the presentdisclosure. The illumination device 756 may include the light source 744and the optic 746 as described for the illumination device 742 of FIG.78A. The light source 744 may comprise a packaged LED that optionallyincludes a lens 758. The lens 758, when present, and the optic 746 maytogether direct emissions from LEDs in a desired emission directionand/or pattern. One or more placement structures 760, such as placementwings of foam and/or silicone, may be provided for ensuring properpositioning within a particular body cavity. The illumination device 756may configured with a power source 762, such as a battery, so that theillumination device 756 may embody a wireless device that does notrequire external electrical connections during use. A circuit element764, such as a flexible circuit, may be provided within the illuminationdevice 756 and an activation baffle 766 may be in communication with thecircuit element 764. A cord 768 may be coupled with the activationbaffle 766 so that when the illumination device 756 is positioned withina body cavity, the cord 768 may be used to activate the activationbaffle 766 and to electrically activate the light source 744. The cord768 may further allow retrieval of the illumination device 756 afterphototherapy is complete. In certain embodiments, the activation baffle766 may comprise silicone. Additionally, the entire device from theactivation baffle 766 to the optic 746 may comprise the protectivecovering 748 as described for FIG. 78A. In other embodiments, theplacement structures 760 may be separately formed around theillumination device 756. The illumination device 756 as described hereinmay be configured as a disposable device with a form factor that issuitable for single use and/or a limited number of uses.

A number of experiments are provided below that demonstrate variousaspects of phototherapy according to principles of the presentdisclosure. The aspects include treatment of human papilloma virus(HPV)-infected tissues for FIGS. 79-81, inhibition of infection and/orreplication of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) for FIGS. 82A-95 , and the efficacy of certain lightagainst wild-type and Tamiflu-resistant influenza A viruses for FIGS.96A-97D.

FIG. 79A-79F is an illustration of an experimental design 770 fortreatment of HPV-infected tissues where an HPV-infected organotypicepithelial raft culture model was used to prepare HPV-infected tissuefor performing anti-viral experiments. For the experiment, HPV-infectedand non-infected organotypic epithelial cultures were provided. As shownin FIG. 79A, primary human keratinocytes (PHKs) that were isolated fromcircumcised neonatal foreskin are subjected to plasmid cotransfection.In FIG. 79B, transfected PHKs are selected based on drug selection andresistance. In FIG. 79C, the transfected PHKs are placed in a dish andcultured with Keratinocyte-Serum Free Medium (K-SFM) on a bed of adermal equivalent. In this case, the dermal equivalent included acollagen support embedded with fibroblasts and held at a medium-airinterface. In FIG. 79D, the transfected PHKs, collagen, and fibroblastsare then transferred to another dish with a mesh support and a raftculture medium. In step FIG. 79E, after greater than 9 days (in thiscase 10 days), the result is a collection of epithelial cellstransfected with HPV-18 that are useful for performing antiviralexperiments. FIG. 79F is a photograph showing HPV-IL L1 viral capsids asdarker image features that are accumulated in the upper live cell strataand cornified layers by D16 in an organotypic raft culture preparedaccording to FIGS. 79A-79F. As shown in the photograph, there arecornified layers, live epithelium, and a dermal equivalent.

As provided by FIGS. 79A-79F, PHKs were isolated from circumcisedneonatal foreskin and grown on a dermal equivalent. After 10 days, thekeratinocytes were stratified and differentiated to form a squamousepithelium HPV-infected cultures transfected with HPV-18 genomicplasmid. HPV-infected and non-infected (or healthy) organotypicepithelial cultures were grown in the dark for six days. Certaincultures were exposed to phototherapy with LED light treatments on days7-12 and harvested on day 13 for characterization. The phototherapyinvolved application of 428 nm light at the following daily-dosages fordifferent culture samples: 12.5 J/cm², or 25 J/cm², or 50 J/cm², or 75J/cm², or 100 J/cm², or 150 J/cm2 daily in either a 10-minute treatmentperiod or with lower irradiance levels over a 7-hour treatment period.Control cultures for both HPV-infected and healthy samples were notexposed to phototherapy. For other cultures, the above daily dosageswere repeated for 7-hour treatment periods.

The following assays were or may be performed on the above-preparedcultures: Histology by Hematoxylin and Eosin, indirectimmunofluorescence detection of BrdU incorporation, Fluorescence In SituHybridization (FISH) to visualize HPV-18 DNA amplification,Immunofluorescence for γ-H2AX, a marker for double-stranded DNA breaks,TUNEL assay to evaluate apoptosis, Quantitative real-time PCR for HPV-18DNA copy number, Immunofluorescence for PCNA, Immunoblot assays: E6, E7,E6AP, Tp53, pRB, p130, g-H2AX. HPV-18 E7-induced host DNA replicationwas abrogated following exposure to 50 J/cm² as indicated by a loss inBrdU positive nuclei. Similar trends were observed following either the10-minute or 7-hour exposures. Similarly, host DNA replication in thebasal strata was abolished following exposure to fluences of 50 J/cm²concluded to be due to the local heating of tissue samples >40° C. fromdrive currents exceeding 2.0 Amps required to deliver the 50 and 75J/cm² doses in a 10-minute window.

One aspect of the present disclosure is that healthy cells and virusinfected cells respond differently to various phototherapy treatments.Particularly, phototherapy differentially impacts the viability ofhealthy and infected cells; the viability of infected cells is retardedmore strongly than the viability of healthy cells. In this regard, FIGS.80A-80J and FIGS. 81A-81J are photomicrographs representing phototherapyexperiments using light units with improved temperature control forHPV-infected and healthy cultures as described above and stained withHematoxylin and Eosin.

FIGS. 80A-80J are photomicrographs of organotypic epithelial cultureswhich were either healthy (FIGS. 80A-80E) or infected with HPV-18 (FIGS.80F-80J) where certain cultures were exposed to phototherapy over a10-minute time period. FIGS. 80A and 80F are healthy and infectedcontrol cultures respectively that were not subjected to phototherapy.FIGS. 80B and 80G were subjected to phototherapy at a dosage of 12.5J/cm², FIGS. 80C and 80H were subjected to phototherapy at a dosage of25 J/cm², FIGS. 80D and 80I were subjected to phototherapy at a dosageof 50 J/cm², and FIGS. 80E and 80J were subjected to phototherapy at adosage of 75 J/cm². It was observed that altered terminaldifferentiation was affected by the phototherapy at 50 J/cm² forinfected cultures (as evidenced by the overlap of keratinized andnon-keratinized cells in FIG. 80I), while this was not observed in thecorrespondingly treated healthy cultures. Highly condensed nuclei, asshown by small dark features in FIGS. 80E and 80J, were evident in bothinfected and healthy cultures at 75 J/cm², indicative of apoptosis. Itwas observed that in healthy cultures, terminal differentiation appearednormal at 50 J/cm² (FIG. 80D) but was negatively affected by thephototherapy at a dose of 75 J/cm² (FIG. 80E) which again led to localtissue heating above 40° C.

FIGS. 81A-81J are photomicrographs of organotypic epithelial cultures(described below in greater detail) which were either healthy (FIGS.81A-81E) or infected with HPV-18 (FIGS. 81F-81J). The respectivecultures illustrated in FIGS. 81A-81E were exposed to the samecumulative dosages as described for FIGS. 80A-80F of phototherapy, butover a 7-hour time frame. For the 7-hour exposure, terminaldifferentiation was observed by the phototherapy starting at 25 J/cm²(FIGS. 81C and 81H) for HPV-18 infected cultures. It was observed thatin healthy cultures, terminal differentiation appeared normal at 50J/cm² (FIG. 81D) but was affected by the phototherapy at 75 J/cm² (FIG.81E). Highly condensed nuclei were evident in both cultures at 75 J/cm²(FIGS. 81E and 81J), indicative of apoptosis. Altered terminaldifferentiation, with overlap of keratinized and non-keratinized cells,is shown in FIG. 81H.

As demonstrated in the photomicrographs of FIGS. 80A-80J and FIGS.81A-81J, altered terminal differentiation may be achieved in infectedcultures before apoptosis of un-infected cultures. This may be evidencedby the observed terminal differentiation starting at 50 J/cm² for the10-minute treatments and at 25 J/cm² for the 7-hour treatments. In boththe 10-minute and 7-hour treatments, apoptosis is indicated at 75 J/cm².In the samples above, host cell DNA replication and proliferation wasdetected with Bromodeoxyuridine (BrdU) immunohistochemistry staining andDNA amplification was detected by HPV-18 fluorescence in situhybridization. The 7-hour and 10-minute exposures were both effective inreducing or abolishing viral activity, with a percent reduction ofHPV-18 DNA copy number/cell of approximately 40% for both 50 J/cm²dosing schedules. In the uninfected PHK cultures, only host cellreplication was observed in the basal or bottom layer of cells. Thisnormal replication was inhibited at 75 J/cm² at both 10-minute and7-hour treatments histologically and confirmed via abundant TUNELpositivity. Based on this, it is observed that some DNA damage may occuras phototherapy dosages increase before apoptosis is realized, thephysiological effects of which are not determined. Cell proliferation inlayers above the basal layer was abnormal, driven by HPV infection. The7-hour exposure at 50 J/cm² was effective in reducing both E6 and E7activities, returning levels similar to normal uninfected raft cultures.Viral protein E7 reactivates cell cycle genes including Cyclin B1 andDNA damage response genes. The DNA damage response proteins stabilizep53, which in HPV-positive cells is normally destabilizing by high riskE6 protein. In these studies, 428 nm blue light results in stabilizationof p53 and reduction of Cyclin B1. Abnormal cell proliferation waseliminated at 75 J/cm², a dose which caused apoptosis in uninfectedcultures. Abnormal cell proliferation was reduced at 25 J/cm² andeliminated at 50 J/cm² for the longer 7-hour treatments. In this regard,potential phototherapy treatment protocols may be realized by inducing abiological effect in infected tissues, e.g., terminal differentiation inthe example of HPV cultures, with applied dosages that provide reducedimpact on healthy tissues.

While not wishing to be bound to a particular theory regarding actionmechanisms, it is believed that blue light may induce oxidative andnitrosative stresses. Referring back to FIG. 75, oxidative stress may becaused by Type 1 and Type 2 reactions. Type 1 reactions can involve theexcitation of an endogenous photosensitizer (most likely an endogenousporphyrin). This excited photosensitizer reacts with another cellularcomponent to form a free radical directly (e.g. superoxide and hydroxylgroups). Type 2 reactions can involve the excitation of an endogenousphotosensitizer (again, most likely a porphyrin) with oxygen to formsinglet oxygen (¹O₂). Nitrosative stress may be induced by the increasein the enzymatic generation of nitric oxide and the photodissociation ofendogenous nitric oxide. Experiments have been performed to show thathigh doses of blue light can increase the expression of nitric oxidesynthase enzymes. The photodissociation of NO from endogenous stores iswell known, with wavelength dependent release of NO fromnitrosated/nitrosylated proteins. HPV infections may be inhibited bynitric oxide and NO-releasing serums may inhibit viral activity and canreduce HPV warts. Literature reports that NO from exogenous sources(e.g., sodium nitroprusside) can inhibit the proliferation and induceapoptosis in high risk HPV cell lines. The more progressed cervicalcarcinomas and HPV-positive dysplastic lesions become, the lessinducible nitric oxide synthase (iNOS) is produced in the tissue. Thismeans that pro-apoptotic concentrations of NO cannot be produced. Lowconcentrations of NO may enhance mutagenesis and increase VEGF-mediatedangiogenesis.

While not wishing to be bound to a particular theory, it is believedthat the phototherapy according to the present disclosure may proceed byone or two different pathways: 1) a nitric oxide pathway, whereincreased NOS expression and photodissociation of NO leads to increasedNO levels, and thus nitrosative stress, and 2) an ROS pathway, whereType I and Type II photoreactions lead to increased ROS levels, whichleads to oxidative stress. In either pathway, inhibited cellproliferation, altered terminal differentiation, DNA damage to hostcells, DNA damage to virus, and apoptosis can be observed. It isproposed that a mechanism of action is that, at increasing phototherapylight doses, infected cultures have DNA damage to host cells, reducedcell proliferation, and altered terminal differentiation. At higherdosages, infected cultures see an inhibition of virus, an eradication ofvirus, and apoptosis of host cells. In non-infected cultures, reducedcell proliferation is observed. At still higher dosages, non-infectedcultures undergo apoptosis. Accordingly, by using phototherapy toincrease NO levels to apoptotic concentrations by increasing free NO andiNOS, one can treat HPV infection. Cells infected with HPV are moresensitive to reactive oxygen species. HPV infected cells are in a stateof chronic oxidative stress. This makes them more susceptible to ROSgenerated by blue light. HPV upregulates E6 protein. The E6 onco-proteinincreases ROS levels in cells and decreases the expression of superoxidedismutase (an enzyme used to mitigate superoxide and convert it to O₂ orH₂O₂).

In another example, aspects are provided in relation to phototherapywith blue light for the inhibition of infection and replication ofSARS-CoV-2. The delivery of safe, visible wavelengths of light can be aneffective, pathogen-agnostic, antiviral therapeutic countermeasure thatwould expand the current portfolio of intervention strategies forSARS-CoV-2 and other respiratory viral infections beyond theconventional approaches of vaccine, antibody, and drug therapeutics.Employing LED arrays, specific wavelengths of visible light may beharnessed for uniform delivery across various targeted biologicalsurfaces. In certain aspects of the present disclosure, it isdemonstrated that primary 3D human tracheal/bronchial-derived epithelialtissues exhibited differential tolerance to light in a wavelength anddose-dependent manner. Primary 3D human tracheal/bronchial tissuestolerated high doses of 425 nm peak wavelength blue light. These studieswere extended to Vero E6 cells to provide understanding of how light mayinfluence viability of a mammalian cell line conventionally used forassaying SARS-CoV-2. Exposure of single-cell monolayers of Vero E6 cellsto similar doses of 425 nm blue light resulted in viabilities that weredependent on dose and cell density. Doses of 425 nm blue light that arewell-tolerated by Vero E6 cells also inhibited SARS-CoV-2 replication bygreater than 99% at 24 hours post-infection after a single five-minutelight exposure. Red light at 625 nm had no effect on SARS-CoV-2replication, or cell viability, indicating that inhibition of SARS-CoV-2replication is specific to the antiviral environment elicited by bluelight. Moreover, 425 nm visible light inactivated up to 99.99% ofcell-free SARS-CoV-2 in a dose-dependent manner. Importantly, doses of425 nm light that dramatically interfere with SARS-CoV-2 infection andreplication are also well-tolerated by primary human 3Dtracheal/bronchial tissue. In this regard, safe, deliverable doses ofvisible light may be considered part of a strategic portfolio fordevelopment of SARS-CoV-2 therapeutic countermeasures to preventcoronavirus disease 2019 (COVID-19).

Among other approaches for treating SARS-CoV-2 infection, there arenucleoside analogs such as Remdesivir, and convalescent plasma, bothseparately demonstrated to shorten time to recovery for Covid-19patients. The glucocorticoid dexamethasone was demonstrated to lower themortality rate in individuals receiving oxygen alone or mechanicalventilation support. To curb the long timelines associated with clinicalsafety and efficacy trials for traditional drug therapeutics,researchers are briskly working to evaluate FDA-approved drugtherapeutics against SARS-CoV-2. Although encouraging, many of thecurrent strategies are SARS-CoV-2 specific and target the virus eitheroutside (cell-free virus), or inside the cell (cell-associated,replicating virus). Expanding the therapeutic armory beyond conventionalstrategies may expedite the availability of therapeutic countermeasureswith non-specific antiviral properties that can inactivate cell-free andcell-associated virus.

Light therapy has the potential to inactivate both cell-free andcell-associated virus. Mitigating SARS-CoV-2 infection with lighttherapy requires knowledge of which wavelengths of light mosteffectively interfere with viral infection and replication, whileminimizing damage to host tissues and cells. A large body of literaturedemonstrates that ultraviolet light, predominantly UVC at the 254 nmwavelength, is highly effective at inactivating cell-free coronaviruseson surfaces, aerosolized, or in liquid. UVC inactivates coronaviruses,as well as many other RNA and DNA viruses, through absorption of UVCphotons by pyrimidines in the RNA backbone, leading to the formation ofpyrimidine dimers that preclude replication of the coronavirus genome.UVC is also highly damaging to replicating mammalian cells, causingperturbations in genomic DNA that can increase the risk of mutagenicevents. As such, viral inactivation with UV light is primarily limitedto cell-free environmental applications. In the present disclosure,inactivating coronaviridae, including coronaviruses, with safe, visiblelight (e.g., above 400 nm) is presented as a new approach to interferingwith SARS-CoV-2 infection and replication.

Photobiomodulation (PBM), or light therapy, is an approach to mitigateoutcomes of viral infection in mammals, such as humans. PBM may alsorefer to phototherapy as disclosed herein. PBM is the safe, low-power,illumination of cells and tissues using light-emitting diodes (LEDs) orlow-level laser therapy (LLLT) within the visible/near-infrared spectrum(400 nm-1050 nm). Importantly, the therapeutic effect is driven bylight's interaction with photoacceptors within the biological system,and is not to be confused with photodynamic therapy (PDT), which employsthe exogenous addition of photosensitizers or chemicals to inducereactive oxygen species (though the addition of photosensitizers orother chemicals to induce reactive oxygen species is another embodimentwithin the scope of the methods described herein).

The safe and effective use of blue light PBM in the 450-490 nm range wasadopted for mainstream clinical use in the late 1960's to treat jaundicein neonates caused by hyperbilirubinemia, and continues to be employedin hospitals today as a primary treatment for hyperbilirubinemia.According to aspects of the present disclosure, changing the wavelengthsof visible light based on targeted applications can broaden the scope oftherapeutic applications. Studies also indicate that PBM with visiblelight may function to inactivate replication of RNA and DNA viruses invitro. Importantly, several studies demonstrate that PBM therapy can besafely applied to the oral and nasal cavities to treat a spectrum ofillnesses. As disclosed herein, PBM therapy in the oral and nasalcavities, as well as in the lungs or endothelial tissues, may be aneffective means of mitigating replication of SARS-CoV-2 in the upperrespiratory tract, so long as it can be done at doses which do notsignificantly affect the viability of the tissues being treated. Adeeper exploration of the precise selection of optical irradiance (e.g.,in mW/cm²) combined with one or more monochromatic wavelengths ofvisible light can broaden the scope of therapeutic applications inrespiratory medicine.

In order to evaluate the safety of visible light on cells and tissues invitro and the efficacy of visible light in SARS-CoV-2 infectious assays,careful designs of LED arrays having narrow band emission spectra withpeak wavelengths at 385 nm, 405 nm, 425 nm, and 625 nm wavelengths areprovided and summarized in FIGS. 82A and 82B. In this manner, LED arraysmay be properly calibrated to provide repeatable and uniform doses oflight so that illumination may occur reliably across many assays and inmultiple laboratories. Measuring the full emission spectrum around thepeak emission wavelength is necessary to confirm proper function foreach LED array and the photon density per nanometer. In this regard,such measurements are recommended as an important characterization stepto help harmonize the variability of results published in literature.FIG. 82A is a chart 772 illustrating measured spectral flux relative towavelength for different exemplary LED arrays. Each LED array wasindependently characterized by measuring the spectral flux, which may bemeasured in W/nm, relative to the wavelength (nm). In FIG. 82A, an LEDarray with a peak wavelength of 385 nm is clearly within the upperbounds of the UVA spectrum (315-400 nm), whereas only a small amount(e.g., about 10%) of an LED array with a peak wavelength of 405 nm lightextends into the UVA spectrum, and an LED array with a peak wavelengthof 425 nm light is 99% within the visible light spectrum (400-700 nm).FIG. 82B illustrates a perspective view of a testing set-up 774 forproviding light from one or more LED arrays 776 to a biological testarticle 778. In addition to the design of the LED arrays 776, includingthe emission spectrums, other important experimental conditionsincluding a distance D of the LED arrays 776 from the biological testarticle 778 (e.g., 90 mm) an illumination power (e.g., 25 mW/cm² or 50mW/cm² depending on the wavelength), and indicated doses (J/cm²) werecarefully calibrated to reduce any effects of temperature on thebiological test articles 778. Moreover, each LED array 776 is validatedto ensure that light is evenly distributed across multi-well tissueculture plates, such that the biological test articles 778 in eachreplicate well receive uniform doses of light.

Understanding how target tissues in the upper airway tolerate blue lightis central to the development of a light-derived antiviral approach forSARS-CoV-2. Initial evaluation of LED arrays was conducted on 3D tissuemodels developed from cells isolated from bronchial/tracheal region of asingle donor. The 3D EpiAirway tissue models are 3-4 cell layers thickcomprising a mucociliary epithelium layer with a ciliated apicalsurface. To assess the wavelength and doses of light most tolerated bythese tissues, replicate tissue samples were exposed to 385 nm, 405 nm,or 425 nm light at various doses. Viability was assayed at 3 hourspost-exposure using the indicated doses and wavelengths of light, anddata is represented as +/−standard deviation. The percent viability oftissue was assessed using a well-established MTT cytotoxicity assayoptimized for the 3D EpiAirway tissue models. FIG. 83A is a chart 780illustrating a percent viability for a peak wavelength of 385 nm fordoses in a range from 0 to 120 J/cm². FIG. 83B is a chart 782illustrating a percent viability for a peak wavelength of 405 nm for thesame doses of FIG. 83A. FIG. 83C is a chart 784 illustrating a percentviability for a peak wavelength of 425 nm for the same doses of FIG.83A. As illustrated in FIGS. 83A-83C, the percent viability of tissuewas clearly impacted in a wavelength-dependent and a dose-dependentmanner. Illumination with 385 nm light exhibited the most dramatic lossin viability with nearly a 50% decrease at a dose of 60 J/cm² (FIG.83A). Light at 385 nm actually showed increased cell viability at dosesof 15 J/cm². Although less dramatic, 405 nm light exhibited adose-dependent decrease in viability with greater than 25% loss at 60J/cm² and about a 50% loss at 120 J/cm² (FIG. 83B). Notably, the 425 nmlight was well tolerated at doses of light out to 120 J/cm² (FIG. 83C).Using 75% viability as a threshold level of acceptable cytotoxicity, 385nm light may be safely administered to these tissues at power levels ofup to 30 J/cm², and 405 nm light may be safely administered to thesetissues at power levels of up to 45 J/cm², and 425 nm light may besafely administered to these tissues at power levels up to 120 J/cm²with only negligible loss of viability between 90 and 120 J/cm², and 425nm doses up to around 75 J/cm² actually showed increased cell viability.

In this regard, 425 nm blue light is shown to have little or no impacton human upper airway-derived 3D tissue models. As such, longerwavelengths of visible light such as 425 nm and greater that do notbleed into the UVA spectrum may have reduced impact on tissue viabilityof primary human tissue derived from the upper respiratory tract. Inparticular, less than 20% tissue loss may be realized at higher doseswith such longer wavelengths. Based on these studies, visible blue lightat 425 nm was chosen for subsequent evaluation in the widely availableVero E6 cell line, conventionally used to evaluate SARS-CoV-2 infectionand replication.

Vero E6 cells are commonly used for preparing stocks, performing growthcurves, and evaluating therapeutic countermeasures for SARS-CoV-2.Depending on the type of assay being performed it could be necessary tovary the seeding cell density and multi-well tissue culture plateformat. Often, cell viability is evaluated to determine if the antiviralproperties of a therapeutic can be parsed from potentialtherapeutic-induced cytotoxic effects. Experiments were performed todetermine if cell density and multi-well plate format can influence cellviability upon exposure to 425 nm blue light. To effectively evaluatethe cell viability, the cytotoxicity assay was optimized for use withVero E6 cell densities up to 1×10⁶ cells.

FIG. 84A is a chart 786 illustrating percent viability for Vero E6 cellsfor antiviral assays performed on 96 well plates at cell seedingdensities of 1×10⁴, 2×10⁴, and 4×10⁴ cells. Under these conditions, itis illustrated that 425 nm blue light may result in decreased cellviability (e.g., 25-50%) at doses of 30 J/cm² and 60 J/cm² by 24 hourspost-illumination, whereas a seeding density of 4×10⁴ cells tolerateshigh doses of light exposure. FIG. 84B is a chart 788 illustratingpercent viability for Vero E6 cells for antiviral assays performed on 48well plates at cell seeding densities of 2×10⁴, 4×10⁴, and 8×10⁴ cells.Unexpectedly, 4×10⁴ cells seeded on a 48-well plate were not welltolerated, showing about a 50% reduction in cell viability at a dose of60 J/cm² compared to 8×10⁴ cells. These results demonstrated that thecell seeding density relative to the surface area of the culture wellinfluences the susceptibility to 425 nm light. FIG. 84C is a chart 790illustrating percent viability for Vero E6 cells for antiviral assaysperformed on 24 well plates at cell seeding densities of 5×10⁴, 1×10⁵,and 2×10⁵ cells. As illustrated, the 24 well plate format of FIG. 84Cwith cell seeding densities of 1×10⁵ and 2×10⁵ demonstrated acceptableviability at all doses tested. In contrast, illumination of Vero E6cells to high doses of 625 nm light may have no impact on cellviability; thereby, indicating that cell density-dependentsusceptibility of Vero E6 cells to 425 nm light appears to becharacteristic of shorter wavelengths of light. Higher Vero E6 seedingdensities resulted in 100% cell confluence prior to illumination,exhibiting cell-to-cell contact that mimics the 3D EpiAirway models.Thus, high confluence Vero E6 cell monolayers readily tolerate 425 nmblue light as well as 3D EpiAirway tissue models.

The use of visible light to inactivate cell-free and cell-associatedcoronaviridae, including coronaviruses, is unprecedented. To assess thecapability of 425 nm blue light to inactivate SARS-CoV-2, Vero E6 cellswere infected with a multiplicity of infection (MOI) of 0.001 SARS-CoV-2isolate USA-WA1/2020 for 1 hour. At 1 hour post-infection (h.p.i.) thecell-associated virus was treated with a single illumination of 425 nmblue light at doses ranging from 7.5 to 60 J/cm². FIG. 85A is a chart792 illustrating tissue culture infectious dose (TCID₅₀) per milliliter(ml) for the 425 nm light at the doses ranging from 7.5 to 60 J/cm² forthe Vero E6 cells infected with a MOI of 0.001 SARS-CoV-2 isolateUSA-WA1/2020 for 1 hour. At 24 h.p.i, there was a clear dose-dependentdecrease in SARS-CoV-2 TCID₅₀/ml. Low doses of 425 nm light weresufficient to reduce SARS-CoV-2 by at least 2 logs for 7.5 J/cm², atleast 3 logs for 15 J/cm², and at least a 5 log reduction for 30 J/cm².A similar trend was observed at 48 h.p.i., although continued viralreplication may account for the similarity in TCID₅₀/ml observed at lowdoses between 7.5 J/cm² and 15 J/cm². This data demonstrates that 425 nmblue light interferes with SARS-CoV-2 replication in a dose-dependentmanner. Specific TCID₅₀/ml values are presented to demonstrate datatrends and data values relative to on another, the actual values mayvary from lab to lab are not meant to be limiting. FIG. 85B is a chart794 illustrating percent reduction in SARS-CoV-2 replication versuspercent cell cytotoxicity for the doses of light as illustrated in FIG.85A. At doses of light that have little impact on the viability of VeroE6 cells (e.g., 7.5, 15, and 30 J/cm²), up to a 99.99% reduction inSARS-CoV-2 replication was observed. Notably, cell viability was a bitlower at 45 J/cm² and 60 J/cm² than the data shown in FIGS. 83A-83C;however, slight variations in the cytotoxicity assay are anticipatedsince the SARS-CoV-2 experiments were executed in independentlaboratories with differences in cell seeding, cell passage, and cellmedia.

FIGS. 86A and 86B represent experimental data similar to FIGS. 85A and85B, but with the MOI increased to 0.01. FIG. 86A is a chart 796illustrating TCID₅₀/ml for 425 nm light at doses ranging from 7.5 to 60J/cm² for Vero E6 cells infected with a MOI of 0.01 SARS-CoV-2 isolateUSA-WA1/2020 for 1 hour. Specific TCID₅₀/ml values are presented todemonstrate data trends and data values relative to on another, theactual values may vary from lab to lab and are not meant to be limiting.FIG. 86B is a chart 798 illustrating percent reduction in SARS-CoV-2replication versus percent cell cytotoxicity for the doses of light asillustrated in FIG. 86A. As illustrated, increasing the MOI to 0.01yielded a similar dose-dependent reduction in SARS-CoV-2 replication aspreviously illustrated for the MOI of 0.001 of FIGS. 85A and 85B.Despite increasing the amount of input virus 10-fold (e.g., from MOI0.001 to MOI 0.01), a short, 2.5 minute dose of 7.5 J/cm² with 425 nmblue light still demonstrated reduction in SARS-CoV-2 replication by atleast 2-logs at 24 h.p.i.

FIG. 86C is a table 800 showing an evaluation of SARS-CoV-2 RNA withreverse transcription polymerase chain reaction (rRT-PCR) for samplescollected for the TCID₅₀ assays of FIGS. 86A-86B. The cycle number fordetection is the basic test result and may be referred to as aquantification cycle (Cq) where low Cq values represent higher initialamount of the target. As shown, there is a dose-dependent reduction inSARS-CoV-2 genomic RNA further substantiating the impact of 425 nm lighton SARS-CoV-2. The fold reduction between doses of 425 nm light withrRT-PCR test detection is lower than those observed for replicationcompetent virus (TCID₅₀ detection), indicating that SARS-CoV-2 viral RNAis readily detectable despite decreases in infectious virions. Thesedata imply that 425 nm blue light may have less of an impact on viralRNA replication and RNA packaging relative to the inactivation of virusparticles.

FIGS. 87A and 87B represent experimental data similar to FIGS. 86A and86B that was obtained by a second, independent laboratory evaluationusing Vero 76 cells infected with a MOI of 0.01 at 48 h.p.i. FIG. 87A isa chart 802 illustrating TCID₅₀/ml for 425 nm light at doses rangingfrom 7.5 to 60 J/cm² for Vero 76 cells infected with a MOI of 0.01SARS-CoV-2. Specific TCID₅₀/ml values are presented to demonstrate datatrends and data values relative to on another, the actual values mayvary from lab to lab and are not meant to be limiting. FIG. 87B is achart 804 illustrating percent reduction in SARS-CoV-2 replicationversus percent cell cytotoxicity for the doses of light as illustratedin FIG. 87A. Consistent with FIGS. 86A and 86B, a similar trend in thedose-dependent effects of 425 nm blue light on SARS-CoV-2 replication isobserved in FIGS. 87A and 87B. Importantly, the dose-dependent trendshowed similar log reductions despite differences in cell type (Vero76), SARS-CoV-2 virus stock preparation, cell culture media, andviability assay.

To understand if the antiviral activity of light against SARS-CoV-2 isspecific to 425 nm blue light, Vero E6 cells infected with a MOI of 0.01were exposed to high doses red light. In this regard, FIG. 88 is a chart806 illustrating TCID₅₀/ml versus various doses of 625 nm red light forVero E6 cells infected with a MOI of 0.01. Specific TCID₅₀/ml values arepresented to demonstrate data trends and data values relative to onanother, the actual values may vary from lab to lab and are not meant tobe limiting. Extensive illumination times with doses ranging from 15J/cm² to 240 J/cm² showed no reduction in TCID₅₀/ml at 24 h.p.i.demonstrating that 425 nm blue light elicits a unique antiviralenvironment that results in SARS-CoV-2 inactivation. In this regard,light at 425 nm can be administered at effective virucidal doses, whichare relatively safe (e.g., less than 25% cytotoxicity) in Vero E6 celllines, and at even higher doses in endothelial cells, like those foundin the respiratory tract and all blood vessels. Red light may havelittle to no effect on SARS-CoV-2 replication, and/or enhances viralload, as measured by TCID₅₀ over 24/48 hours. However, red light maydecrease inflammation resulting from exposure to blue light, which maypositively impact cell viability, thereby lowering cytotoxicity. Adecrease in severe inflammatory response can be beneficial when treatingviral infections, particularly when a virus can elicit a cytokine stormand/or inflammation can result in secondary bacterial infections.Accordingly, the combination of blue light, such as light at around 425nm, and red light at one or more anti-inflammatory wavelengths, canprovide a desirable combination of biological effects.

Efficacy of 425 nm blue light against cell-associated SARS-CoV-2 can bea combination of blue light eliciting an antiviral environment in thecells and inactivating cell-free virions. To distinguish between these,FIGS. 89A and 89B represent cell-free SARS-CoV-2 inactivation that wasevaluated by two independent laboratories. Two different virussuspensions containing the equivalent of ˜10⁵ and ˜10⁶TCID₅₀/ml wereilluminated with the indicated doses of 425 nm blue light. Followingillumination, virus was assayed by TCID₅₀ on Vero E6 cells in a firstlaboratory as illustrated in a chart 808 of FIG. 89A and on Vero 76cells in a second laboratory as illustrated in a chart 810 of FIG. 89B.As illustrated in FIG. 89A, in the first laboratory, low doses of 425 nmlight were sufficient to inactivate 10⁶TCID₅₀/ml SARS-CoV-2 with atleast 1 log reduction at 7.5 J/cm² (or greater than 90%), with at least2 log reduction at 15 J/cm² (or greater than 99%), with at least 3 logreduction at 30 J/cm² (or greater than 99.9%), and at least 4 logreduction at 60 J/cm² (or greater than 99.99%). A similar trend in thedata was observed in the second laboratory for the Vero 76 cells asillustrated in FIG. 89B. Despite a less dramatic reduction in SARS-CoV-2inactivation, at least a 2 log reduction was still observed at 60 J/cm²(or at least 99%). Technical differences between laboratories includingSARS-CoV-2 virus stock preparation, cell culture media, and cell typesused for assaying virus may be factors that influenced the magnitude ofsusceptibility. Overall, the results from two independent laboratoriesdemonstrated that low doses of 425 nm blue light (e.g., 15 J/cm²)effectively inhibits the infection and replication of cell-free andcell-associated SARS-CoV-2, with minimal impact on cell viability.Specific TCID₅₀/ml values are presented to demonstrate data trends anddata values relative to on another, the actual values may vary from labto lab and are not meant to be limiting.

For completeness of collected data, FIGS. 90A and 90B are provided toshow that Vero E6 cells do not exhibit decreased percent viability whenexposed to doses of green light or doses of red light. In both FIGS. 90Aand 90B, a number of cells was provided at 2×10⁵ cells, 1×10⁵ cells, and5×10⁴ cells. FIG. 90A is a chart 812 indicating that Vero E6 cells donot show decreased viability under 530 nm light at doses ranging from0-180 J/cm². FIG. 90B is a chart 814 indicating that Vero E6 cells donot show decreased viability under 625 nm light at doses ranging from0-240 J/cm².

The expedited need for therapeutic countermeasures against SARS-CoV-2and other respiratory viral pathogens beckons the rapid development ofnovel approaches that may complement existing public health measures. Asdisclosed herein, LED arrays were carefully designed to demonstrate forthe first time that safe, visible blue 425 nm light can inhibit bothcell-free and cell-associated SARS-CoV-2 infection and replication in adose-dependent manner. Results from two independent laboratoriesdemonstrate that low doses of 425 nm blue light (e.g., 15 J/cm²)effectively inhibit infection and replication of SARS-CoV-2(e.g., >99%), with minimal impact on Vero E6 cell viability.Importantly, doses of 425 nm light 60 J/cm² were well tolerated in the3D EpiAirway tissue models established from human tracheal/bronchialtissues.

The EpiAirway model is a commercially available in vitro organotypicmodel of human mucociliary airway epithelium cultured at the air/liquidinterface to provide a differentiated in vivo-like epithelial structurewith barrier properties and metabolic functions. There is strong globalmomentum to replace animal model testing with relevant in vitrohuman-derived test systems to reduce the number of animals used inpreclinical testing. Current testing guidelines (TG403, TG433, andTG436), established by the Organization for Economic Co-operation andDevelopment (OECD), for inhalation toxicity outline the use of animalsto determine LC₅₀ (e.g., a concentration required to cause death of 50%of the test animals). The EpiAirway in vitro tissue model can be used todetermine the IC₂₅ value (concentration required to reduce tissueviability by 25% of vehicle control-treated tissues) of a test article.Following 3 hours of exposure, the model have been shown to predictrespiratory tissue viability using chemicals with the GloballyHarmonized System (GHS) Acute Inhalation Toxicity Category 1 and 2, andEnvironmental Protection Agency (EPA) Acute Inhalation Toxicity CategoryI-II classifications. Extended exposure times (e.g., 24 and 72 hours)with toxic chemicals also reflect in vivo responses and havedemonstrated the predictive value of the EpiAirway models forrespiratory toxins in humans. Furthermore, such a uniform in vitro modelis ideally suited to evaluate the safety doses of light applied to afixed surface area (e.g., in J/cm²), rather than attempting to scale theoptical delivery of light to the appropriate small rodent anatomy.

As previously shown in FIGS. 83A-83C, the EpiAirway model was exposed tovarious dose ranges at light with wavelengths of 385 nm, 405 nm, and 425nm. Exposure to UVA light at 385 nm exhibited greater than 25% loss inviability at greater than 45 J/cm², identifying a dose that breaches theIC₂₅ threshold established for acute cytotoxicity in the EpiAirwaymodel. In contrast, higher doses of the 425 nm blue light reached theIC₂₅ threshold for validated acute airway irritation. Greater than 100%tissue viability was observed following illumination with antiviral(e.g., >99.99% reduction in SARS-CoV-2) 425 nm blue light doses of 60J/cm². The distinct viability profiles observed at 385 nm, 405 nm, and425 nm demonstrate that the 3D EpiAirway tissue models are amenable foridentifying acute respiratory effects associated with light therapy in adose- and wavelength-dependent manner. Minimal loss in viability out to120 J/cm² at 425 nm indicates that the 3D human respiratory tissuemodels are highly tolerant to this wavelength. In FIGS. 84A to 84C, 2DVero E6 cell cultures exhibited a cell density-dependent viabilityresponse to 425 nm doses at greater than or equal to 15 J/cm², whereinlow seeding densities per surface area were more susceptible tolight-induced cytotoxic effects. The enhanced tolerance of the 3DEpiAirway tissue models to 425 nm blue light compared to 2D Vero E6 cellcultures is not surprising, given that cells in 3D culture are oftenmore resistant to drug treatment, drug metabolism is more effective, andthere is increased resistance to drug-induced apoptosis. Thecharacteristics of 3D tissue models more closely reflect cellularattributes observed in the context of tissues in vivo. Developingoptimal conditions for SARS-CoV-2 infection and replication in 3Drespiratory tissue models will help elucidate mechanisms that govern theability of 425 nm blue light to inactivate SARS-CoV-2.

The mechanisms underlying 425 nm blue light to inactivate SARS-CoV-2 arestill being developed; however, a brief introduction to putativemolecular contributors is relevant. The molecular mechanisms governingthe impact of blue light on non-pigmented cells are only beginning to berevealed. The effects of blue light should follow the first law ofphotochemistry, which states that light must be absorbed to have aneffect. A handful of photoacceptors for blue light have been identifiedin non-pigmented cells, including cytochrome c oxidase, flavins,porphyrins, opsins, and nitrosated proteins. Light absorption byphotoreceptors can lead to release of reactive oxygen species (ROS)and/or nitric oxide (NO) that may function to inactivate SARS-CoV-2 in acell-free or cell-associated environment. Reactive oxygen species and/orbioactive NO may elicit activation of transcription factors involved inimmune signaling, such as nuclear factor kappa-light-chain-enhancer ofactivated B cells (NF-KB) and mitogen activated protein kinase (MAPK)signaling. NFKB and MAPK pathways can lead to transcriptional activationof innate and inflammatory immune response molecules that may interferewith SARS-CoV-2 replication. Nitric oxide may also mediate inactivationof cell-associated SARS-CoV-2 through S-nitrosylation of cysteineresidues in the active site of viral encoded enzymatic proteins.Reactive oxygen species and/or NO may also function to inactivatecell-free virions. Photosensitizers present in cell media may facilitategeneration of ROS and/or NO that directly impact virion proteins and/orviral RNA to prevent infection and replication. It has also beendemonstrated that inactivation of cell-free feline calicivirus (FCV) by405 nm light was dependent on naturally occurring photosensitizers inmedia. Importantly, FCV was inactivated by 4 logs in artificial salivaand blood plasma, indicating that light-induced inactivation ofcell-free virus is obtainable under biologically-relevant conditions.Evidence demonstrating that SARS-CoV-2 can be inactivated by exogenousaddition of NO donor molecules, or possibly by single oxygen,substantiates the potential for SARS-CoV-2 inactivation by nitric oxide.

In the above described experiments, materials and methods are providedin more detail below for reference. With regard to cells, tissues, andviruses, Vero E6 cells were purchased from ATCC and maintained in DMEM(Sigma-Aldrich) supplemented with 10% FetalClonell (HyClone) and 1%Antibiotic-Antimycotic (Gibco). Vero 76 cells (ATCC CRL-1587) weremaintained in MEM supplemented with 2 mM L-glutamine and 5% FBS. Primaryhuman airway epithelium (EpiAirway AIR-100, MatTek Corporation) werecultured for 28 days in transwell inserts by MatTek Corporation. Thecultured tissues were shipped in 24 well plates with agarose embedded inthe basal compartment. Upon arrival, the transwell inserts were removedand placed in 6-well plates with cold Maintenance Media in the basalcompartment; no media added to the apical surface. Cells were incubatedat 37° C. and 5% CO₂ overnight prior to experimental use. All work withlive virus was conducted in two independent Biosafety Level-3 (BSL-3)laboratories, MRI Global's Kansas City facility and the Institute forAntiviral Research at Utah State University, with adherence toestablished safety guidelines. At both laboratories, SARS-CoV-2(USA-WA1/2020) was obtained from the World Reference Center for EmergingViruses and Arboviruses (WRCEVA) and propagated with slightmodifications. At MRI Global, Vero E6 cells were cultured overnight withDMEM (Gibco; 12320-032) supplemented with 10% FBS (Avantor, 97068-085),1% nonessential amino acids (Corning 25-025-CI), and 1%penicillin/streptomycin (VWR 97063-708). To generate master stocks,cells were infected prior to infection with an approximate MOI of 0.08in infection media (as above with 5% FBS). Cells were monitored forcytopathic effects daily and harvested at 4 days post-infection as CPEapproached 100%. Working stocks were cultured in Vero E6 cells withDMEM/F12 media (Gibco; 11330-032) supplemented with 10% FBS and 1%penicillin/streptomycin at an MOI of 0.005. Cells were monitored for CPEand harvested two days post-infection as CPE approached 70%. Cellculture debris was pelleted by centrifugation at 500×g for 5 min andviral stocks were stored at −80° C. Infectivity of viral stocks wasdetermined by TCID50 assay. At Utah State University, SARS-CoV-2(USA-WA1/2020) was propagated in Vero 76 cells. Infection media wasMinimal Essential Media supplemented with 2 mM I-glutamine, 2% FBS, and50 μg/mL gentamicin.

For cytotoxicity assays for human tissues, prior to illumination, themaintenance media was changed on the human tissue transwell inserts.Tissues were illuminated with 385 nm, 405 nm, or 425 nm light andincubated at 37° C. and 5% CO₂ for 3 hours. Cytotoxicity was determinedusing the EpiAirway MTT assay following manufacturer's instructions.Briefly, tissues were rinsed with TEER buffer and placed into pre-warmedMTT reagent and incubated at 37° C. and 5% CO₂ for 90 minutes. The MTTsolution was extracted with MTT extractant solution by shaking for 2hours. The tissue inserts were discarded and the extractant solution wasadded to a 96 well plate to be read at 570 nm. Extractant solutionserved as the experimental blank and cell viabilities were calculatedagainst plates that were not illuminated.

For cytotoxicity assays for cell lines, Vero E6 cells were incubated inclear 24-well, 48-well, and 96-well plates (Corning) at varying seedingdensities and incubated at 37oC and 5% CO₂ overnight. Cells wereilluminated with 385 nm, 405 nm, or 425 nm light and incubated at 37° C.and 5% CO₂ for 24 hours post-illumination. After 24 hours, cytotoxicitywas determined using the CellTiterGlo One Solution (Promega) withmodifications. The amount of CellTiterGlo One Solution (“CTG”) wasoptimized in a preliminary experiment. For 24-well plates, 100 μlsolution was used and 60 μl solution was used for 48- and 96-wellplates. The cells were placed on an orbital shaker for 2 minutes and thechemiluminescent signal was stabilized for 10 minutes before 50 μl ofthe solution was added to a black well, black bottom 96-well plates andread using the CellTiterGlo program on the GloMax (Promega).CellTiterGlo One solution served as a blank and cell viabilities werecalculated against plates that were not illuminated.

Cytotoxicity analysis was conducted at 48 hours post-illumination. Cellswere treated for 2 hours with 0.01% neutral red for cytotoxicity. Excessdye was rinsed from cells with PBS. Absorbed dye was eluted from thecells with 50% Sorensen's citrate buffer/50% ethanol for 30 minutes.Buffer was added to 10 wells per replicate. Optical density was measuredat 560 nm and cell viabilities were calculated against cells that werenot illuminated.

Antiviral assays were conducted in separate laboratories withmodifications. At MRI Global, cells were infected with SARS-CoV-2 atMOIs of 0.01 and 0.001 in triplicate. At one hour post-infection,infected cells were illuminated with 425 nm light at the specifieddoses. Cell culture supernatants were harvested at 24 hours and 48 hourspost-infection for TCID₅₀ determination and qPCR analysis. Noillumination controls and no virus controls were included as a positivecontrol for viral growth and for cytotoxicity, respectively.Cytotoxicity analysis was conducted at 24 hours post-illumination asabove.

Vero 76 cells were infected with SARS-CoV-2 at MOIs of 0.01 and 0.001.At one hour post-infection, infected cells were illuminated with 425 nmlight at the specified doses. Cell culture supernatants were harvestedat 48 hours post-infection for TCID₅₀ determination. No illuminationcontrols and no virus controls served as a positive control for viralgrowth and for cytotoxicity, respectively. Cytotoxicity analysis wasconducted at 48 hours post-illumination.

Virucidal assays were conducted in parallel in separate laboratories. Atone laboratory, 1 mL solutions containing 10⁵ and 10⁶ TCID₅₀/ml wereilluminated with varying doses of light. The viruses were then titteredon Vero E6 cells in triplicate via TCID₅₀ assay. No illuminationcontrols served as a positive control for viral growth.

At a second laboratory, 1 mL solutions containing 10⁵ and 10⁶ TCID₅₀/mlwere illuminated with varying doses of light. The viruses were thentittered on Vero 76 cells in triplicate via TCID₅₀ assay. Noillumination controls served as a positive control for viral growth.

Viral RNA levels for SARS-CoV-2 samples were determined by quantitativeRT-PCR using the CDC N1 assay. Samples for the RT-PCR reactions werelive virus in culture supernatants without nucleic acid extraction.Primers and probes for the N1 nucleocapsid gene target region weresourced from Integrated DNA Technologies (2019-nCoV CDC RUO Kit, No.10006713). TaqPath 1-step RT-qPCR Master Mix, CG was sourced fromThermoFisher (No. A15299). Reaction volumes and thermal cyclingparameters followed those published in the CDC 2019-Novel Coronavirus(2019-nCoV) Real-Time RT-PCR Diagnostic Panel: Instructions for Use. Forthe RT-PCR reaction, 15 mL of prepared master mix was added to each wellfollowed by 5 mL of each sample, for a final total volume of 20 mL perreaction well. Reactions were run on a Bio-rad CFX real-time PCRinstrument.

TCID50 assays were conducted as follows at both laboratories with slightmodifications. At one laboratory, Vero E6 cells were plated in 96-wellplates at 10,000 cells/well in 0.1 ml/well of complete medium (DMEM/F12with 10% fetal bovine serum and 1X Penicillin/Streptomycin) andincubated overnight in a 37° C., 5% CO₂ humidified incubator. The nextday virus samples were serially diluted into un-supplemented DMEM/F12media at 1:10 dilutions by adding 0.1 ml virus to 0.9 ml diluent,vortexing briefly and repeating until the desired number of dilutionswas achieved. Media was decanted from 96-well plates and 0.1 ml of eachvirus dilution aliquoted into 5 or 8 wells. After 4 days of incubationat 37° C., 5% CO₂, plates were scored for presence of cytopathic effect.TCID₅₀/ml were made using the Reed & Muench method. At the secondlaboratory, cell culture samples were serially diluted and plated onfresh Vero 76 cells in quadruplicate. Plates were visually examined forCPE at 6 days post-infection. Wells were indicated as positive ornegative and virus titers were calculated using the Reed-Muench endpointdilution method.

FIG. 91A is a chart 816 showing raw luminescence values (RLU) fordifferent seedings of Vero E6 cell densities and various doses of light(J/cm²). FIG. 91B is a chart 818 showing percent viability for thedifferent seedings of Vero E6 cell densities and various doses of lightof FIG. 91A. FIG. 91B indicates the viability of Vero E6 cells may notreach saturation until cell densities are above 10⁶ cells. RLU andpercent viability based on the various doses of light demonstrate thatboth 100 μL and 200 μL of CellTiter-Glo (CTG) are effective volumes formeasuring cell viability after seeding different Vero E6 cell densities.For FIGS. 91A and 91B, cell densities of 2×10⁵ cells with 100 μL CTG,1×10⁵ cells with 100 μL CTG, 5×10⁴ cells with 100 μL CTG, 2×10⁵ cellswith 200 μL CTG, 1×10⁵ cells with 200 μL CTG, and 5×10⁴ cells with 200μL CTG are represented. FIG. 91C is a chart 820 comparing RLU versustotal cell number to show that CTG is an effective reagent for measuringcell densities of above 10⁶ Vero E6 cells. RLU values versus total cellnumber are provided for 500 μL CTG, 250 μL CTG, and 100 μL CTG and datais represented as +/−standard deviation.

FIG. 92A is a chart 822 of TCID₅₀/ml versus dose at 24 hours and 48hours post infection for Calu-3 cells infected with SARS-CoV-2. SpecificTCID₅₀/ml values are presented to demonstrate data trends and datavalues relative to on another, the actual values may vary from lab tolab and are not meant to be limiting. FIG. 92B is a chart 824 showingthe percent reduction in SARS-Cov-2 compared with percent cytotoxicity,for the Calu-3 cells of FIG. 92A. For FIG. 92B, the chart lines forpercent reduction in SARS-Cov-2 and percent cytotoxicity are provided asnonlinear regression curves based on the doses illustrated in FIG. 92A.As shown, visible light at 425 nm inhibits viral replication ofSARS-CoV-2 in the human respiratory cell line, Calu-3. The Calu-3 cellswere infected with SARS-CoV-2 at an MOI of 0.1 and exposed to theindicated doses of 425 nm light at 1 hour post-infection. SARS-CoV-2samples were harvested for TCID₅₀ assays at 24 hours and 48 hourspost-infection. Greater than a 99% reduction in virus was observedfollowing a single treatment for doses of 15 J/cm². Percent reduction inSARS-CoV-2 virus as shown in FIG. 92B was calculated for each dose andtimepoint. As previously described, the selectivity index (SI) may bedefined as a ratio of the CC₅₀ to the EC₅₀ for treated cells. As shownin FIG. 92B, 50% percent reduction in SARS-CoV-2 at 24 hours and 48hours post infection are indicated at relatively low dose values. Inthis regard, the doses of light that inhibit viral replication havedesirable SI values of greater than 100 24 hours post infection andgreater than 25 when factoring in the cell viability of Calu-3 cells notinfected with virus.

FIG. 93A is a chart 826 illustrating percent reduction in SARS-CoV-2replication versus percent cell cytotoxicity for Vero E6 cells infectedwith a MOI of 0.01. FIG. 93B is a chart 828 illustrating percentreduction in SARS-CoV-2 replication versus percent cell cytotoxicity forVero E6 cells infected with a MOI of 0.001. In both FIGS. 93A and 93B,the indicated doses were applied at 1 hour post infection and doseresponses were determined at 24 hours post infection. The doses wereadministered by application of 425 nm light with an irradiance of 50mW/cm² for times of 2.5 minutes (for 7.5 J/cm²), 5 minutes (for 15J/cm²), 10 minutes (for 30 J/cm²), 15 minutes (for 45 J/cm²), and 20mins (for 60 J/cm²). Consistent with previously presented charts,similar trends are observed for dose-dependent effects of 425 nm bluelight on SARS-CoV-2 replication for both MOI values. The cytotoxicitycurve indicates a CC₅₀ of about 30.2. In FIG. 93A, the percent reductionin SARS-CoV-2 is close to 100% for doses as low as 7.5 J/cm² and thecorresponding nonlinear regression curve has a sharp decrease at or nearthe 0 J/cm² dose. For the purposes of SI calculations, a conservativevalue of 1 was selected for the EC₅₀ value to give a SI value (e.g.,CC₅₀/EC₅₀) of about 30. In FIG. 93B, the percent reduction in SARS-CoV-2is farther away from 100% for the 7.5 J/cm² dose, thereby providing thecorresponding nonlinear regression curve with a decrease toward 0% at adose slightly above the 0 J/cm² dose. In this manner, a value of about3.4 may be indicated for the EC₅₀ value to give a SI value (e.g.,CC₅₀/EC₅₀) of about 9. Due to variability in experiments, slightdifferences in data sets may be expected. In this regard, the resultsillustrated in FIGS. 93A and 93B may be considered as similar and withinnormal experimental variations.

While FIGS. 93A and 93B provide percent reduction in SARS-CoV-2 at thecellular level for determining EC₅₀ values, IC₂₅ values for targettissues are needed to determine suitable LTI treatment values. FIG. 93Cis a chart 830 representing percent viability at various doses forprimary human tracheal/bronchial tissue from a single donor. Tissueviability is determined at 3 hours post-exposure by MTT assay, a measureof cell viability by assessing enzymatic activity of NAD(P)H-dependentcellular oxidoreductase ability to reduce MTT dye to formazan. From thechart 830, the IC₂₅ value corresponds to the dose where the viabilitycurve is at 75% (e.g., 25% reduction in tissue viability). In FIG. 93C,the IC₂₅ value is about 157, as indicated by the superimposed dashedlines. In combination with the EC₅₀ values of FIGS. 93A and 93B, thecorresponding LTI values may be determined as about 157 for FIG. 93A andabout 46 for FIG. 93B.

FIGS. 94A-94C repeat the experiments of FIGS. 93A-93C, but with lighthaving a peak wavelength of 450 nm. FIG. 94A is a chart 832 illustratingpercent reduction in SARS-CoV-2 replication versus percent cellcytotoxicity for Vero E6 cells infected with a MOI of 0.01. FIG. 94B isa chart 834 illustrating percent reduction in SARS-CoV-2 replicationversus percent cell cytotoxicity for Vero E6 cells infected with a MOIof 0.001. Consistent with previously presented charts, similar trendsare observed for dose-dependent effects of 450 nm blue light onSARS-CoV-2 replication for both MOI values. The cytotoxicity curvesindicate a CC₅₀ of greater than 60 since the curve does not extend to50% cytotoxicity. In turn, SI values based on CC₅₀ value of greater than60 may also be considered as greater than the particular SI values. InFIG. 94A, a value of about 7.2 may be indicated for the EC₅₀ value togive a SI value (e.g., CC₅₀/EC₅₀) of greater than 8. In FIG. 94B, avalue of about 4.1 may be indicated for the EC₅₀ value to give a SIvalue (e.g., CC₅₀/EC₅₀) of about greater than 15. As before, due tovariability in experiments, slight differences in data sets may beexpected. In this regard, the results illustrated in FIGS. 94A and 94Bmay be considered as similar and within normal experimental variations.Notably, Vero E6 cells receiving similar doses of 450 nm blue lightexhibit less cytotoxicity than that observed for 425 nm blue light,demonstrating a wavelength-dependent biological effect.

FIG. 94C is a chart 836 representing percent viability at various dosesfor primary human tracheal/bronchial tissue from a single donor. As withFIG. 93C, tissue viability is determined at 3 hours post-exposure by MTTassay. From the chart 836, the IC₂₅ value may be determined at about330. In combination with the EC₅₀ values of FIGS. 94A and 94B, thecorresponding LTI values may be determined as about 46 for FIG. 94A andabout 80 for FIG. 94B. While FIG. 94C shows about 63% viability at adose of 360 J/cm², variability between biological replicates was high atthis dose. In this regard, the IC₂₅ values may be even greater than theapproximated value of 330, indicating very high doses may beadministered before significant toxicity is observed. Consistent withdata in Vero E6 cells, primary human respiratory tissues receiving 450nm blue light exhibit an increased tolerance to higher doses compared to425 nm blue light, demonstrating a wavelength-dependent biologicaleffect.

FIG. 95 is a table 838 summarizing the experiments of FIGS. 93A-93C and94A-94C. The higher SI and LTI values for 450 nm light are predominantlya consequence of lower cytotoxicity relative to 425 nm light. Lower EC₅₀values demonstrate more effective virus inhibition at 425 nm, but thiscan be associated with higher cytotoxicity values at lower light dosesthan at 450 nm. Ideally, light therapy may include lower EC₅₀ valueswith CC₅₀ values as high as possible. Different targeted pathogens andtissue types may provide different LTI values. In this regard, LTIvalues according to the present disclosure may be provided at values ofgreater than or equal to 2, or in a range from 2 to 100,000, or in arange from 2 to 1000, or in a range from 2 to 250, depending on theapplication. Considering experimental variances, the exemplary dataprovided for treatment of SARS-CoV-2 with light in a range from 425 nmto 450 nm indicates LTI values in any of the above ranges may beachieved.

Using techniques analogous to those used above to measure the antiviralactivity of 425 nm to 450 nm light against SARS-CoV-2, the antiviralactivity of light at 405 nm to 425 nm against wild-type (WT) andTamiflu-resistant influenza A was investigated. FIG. 96A is a chart 840showing the titer of WT influenza A virus based on remaining viral loadsfor different initial viral doses after treatment with different dosesof 405 nm light. The initial viral doses were set at 1×10⁴ and 1×10⁵,and the remaining viral load (e.g., TCID₅₀/ml) following treatment withlight at 405 nm at dosages of 0 J/cm², 60 J/cm², and 120 J/cm² is shown.The data demonstrates significant reductions in wild-type influenza Aviral loads when either 60 J/cm² or 120 J/cm² doses were administered,with an additional roughly 0.5-log reduction in viral loads observed atthe higher dosage.

FIG. 96B is a chart 842 showing the titer of Tamiflu-resistant influenzaA virus based on remaining viral load for a single initial viral doseafter treatment of different doses of 425 nm light. The initial viraldose was set at 1×10⁴, the remaining viral load (e.g., number of copies)following treatment with light at 425 nm at dosages of 0 J/cm², 60J/cm², and 120 J/cm² is shown. The initial dose is provided at about1×10⁴, and the remaining viral load (e.g., number of copies) followingtreatment with light at 425 nm at dosed of 0 J/cm², 30 J/cm², 60 J/cm²,120 J/cm², 180 J/cm², and 240 J/cm² is shown. The data shows an increasein viral load when no light was administered, and dose-dependentreductions in viral loads up to about 180 J/cm², totaling a roughly2-log reduction in viral load.

FIG. 97A is a chart 844 showing the TCID₅₀/ml versus energy dose for WTinfluenza A treated with light at 425 nm at various doses. The MOI forthe WT influenza A was provided at 0.01. The selected doses wereprovided at 0 J/cm², 3 J/cm², 7.5 J/cm², 15 J/cm², 30 J/cm², 45 J/cm²,60 J/cm² and 90 J/cm². Results were collected after 24 hours and after48 hours. When no light was applied (e.g., dose of 0 J/cm²), viral loadsincreased to 10³ copies at 24 hours, and to 10⁵ copies at 48 hours. Atdoses between about 7.5 J/cm² and 60 J/cm², a dose-dependent decrease inviral loads was observed at 24 hours, though the virus significantlyrebounded by 48 hours. However, at doses of 90 J/cm², the viral loadssignificantly decreased by 24 hours, and did not significantly increaseat 48 hours. Specific TCID₅₀/ml values are presented to demonstrate datatrends and data values relative to on another, the actual values mayvary from lab to lab and are not meant to be limiting.

FIG. 97B is a chart 846 showing the percent reduction in viral loads ofWT influenza A and percent cytotoxicity against the treated cells wheninfluenza A-infected Madin-Darby Canine Kidney (MDCK) cells were exposedto 425 nm light at various doses. The MOI for the WT influenza A wasprovided at 0.01. As illustrated, the doses were provided at 0 J/cm²,7.5 J/cm², 15 J/cm², 30 J/cm², 45 J/cm², 60 J/cm² and 90 J/cm². Thereduction in viral loads and the cytotoxicity were monitored at 24 and48 hours post irradiation. Virtually no cytotoxicity was observed at anytime period for any of the doses. The reduction in viral loads was dosedependent, with doses of 45 J/cm², 60 J/cm², and 90 J/cm² demonstratinga nearly complete reduction in viral loads.

FIG. 97C is a chart 848 that is similar to FIG. 97A, but with a startingMOI of 0.1. In this regard, FIG. 97C illustrates the TCID₅₀ of cellsinfected with WT influenza A and treated with 425 nm light at doses of 0J/cm², 3 J/cm², 7.5 J/cm², 15 J/cm², 30 J/cm², 45 J/cm², 60 J/cm² and 90J/cm². Results were collected after 24 hours and after 48 hours. Viralloads stayed fairly constant at 24 hours for doses from 0 to 15 J/cm²and decreased in a dose dependent manner as the doses increased to 90J/cm². Over the next 24 hours (i.e., a total of 48 hours post-exposure),the viral loads significantly rebounded at all dosages other than 90J/cm².

FIG. 97D is a chart 850 that is similar to FIG. 97B, but with a startingMOI of 0.1. In this regard, FIG. 97D illustrates the percent reductionin viral loads of WT influenza A and percent cytotoxicity against thetreated cells when influenza A-infected Madin-Darby Canine Kidney (MDCK)cells were exposed to 425 nm light at various doses. The MOI for the WTinfluenza A was provided at 0.1. As illustrated, the doses were providedat 0 J/cm², 7.5 J/cm², 15 J/cm², 30 J/cm², 45 J/cm², 60 J/cm² and 90J/cm². The reduction in viral loads and the cytotoxicity were monitoredat 24 and 48 hours post irradiation. As with FIG. 97B, virtually nocytotoxicity was observed at any time period for any of the doses andthe reduction in viral loads was dose dependent, with doses of 45 J/cm²,60 J/cm² and 90 J/cm² demonstrating a high or nearly complete reductionin viral loads. Specific TCID₅₀/ml values are presented to demonstratedata trends and data values relative to on another, the actual valuesmay vary from lab to lab and are not meant to be limiting.

As a summary of the findings, therapeutic light treatments can beselected from optimal doses including various combinations ofwavelengths, irradiance, and treatment times as discussed above forvarious viruses, including coronaviridae (e.g., coronavirus, SARS-CoV-2,etc.) and Orthomyxoviridae (e.g., influenza), among others. Ideally, thephototherapy may induce a dual mechanism of action on the virus,including damaging the lipid membrane using single oxygen and/or nitricoxide. The treatments demonstrate efficacy both extracellular in theabsence of cells pre-infection, as well as intracellular in the presenceof cells post infection. The antiviral effect can be remarkably fast.For example, inactivation of the SARS-CoV-2 virus was demonstratedwithin 24 to 48 hours, compared to the course of viral load reductionobserved clinically as the SARS-CoV-2 virus clears the body in untreatedpatients, or even in patients treated with Remdesivir.

It is important to consider the “Light Therapeutic Index,” or “LTI,” aratio of the IC₂₅ and the EC₅₀ values for light that is used on cellsand tissues. Ideally, the light treatment will be effective at killingone or more target viruses at power levels that are not overlycytotoxic. Preferably, the ratio of IC₂₅/EC₅₀ is as high as possible,including greater than 2. Cell systems for each virus have a number ofvariables (e.g., cell density, different cell types for productiveinfection, media, etc.), which makes it hard to have a single LTI forall viruses. Important aspects for evaluating LTI across all viruses,particularly for respiratory viruses, include evaluating the types ofhuman tissue these viruses are likely to infect, such as EpiAirway fromboth large airway (AIR-100) and nasal (NAS-100) tissues. EpiAirway is aready-to-use, 3D mucociliary tissue model consisting of normal,human-derived tracheal/bronchial epithelial cells, also available as aco-culture system with normal human stromal fibroblasts (EpiAirwayFT). Areduction as large as 75-fold is observed after a 2.5-minute treatmentdose at 50 mW/cm². The light therapy shows significant antiviralactivity post infection, inhibiting about 50% of viral replication.Additionally, this treatment shows a full log inactivation of virus onWT influenza A at doses of greater than 8.5 J/cm². A dose of 8.5 J/cm²was a dose that provided an EC₅₀ against influenza post infection. Inthis regard, doses of less than 10 J/cm² can provide a multi-pathogenictreatment that can eliminate different viruses via one or more separatemechanisms. In a particular example, a multi-pathogenic treatment of 425nm light for 5 minutes and an irradiance of 50 mW/cm² may be effectivefor treating both SARS-CoV-2 and influenza A. Additionally, at doses ofaround 60 J/cm², a greater than 2-log reduction in virucidal activitywas observed using 425 nm light with a 20-minute exposure at 50 mW/cm².

Considering LTI calculations (e.g., the ratio of IC₂₅/EC₅₀) in antiviralassays for specific tissues for SARS-CoV-2 and influenza at just 425 nm,it is observed that there are safe and effective doses of light that canbe administered. Because the viral lipid membranes are similar for otherrespiratory viruses, it is believed (based on successful results withSARS-CoV-2 and influenza A) that such treatments can be effectiveagainst other respiratory viruses. When comparing the results with lightat 425 nm with the results at 405 nm or 385 nm, the LTI may be smaller,though it will be expected to vary depending on tissue types.Extrapolating the data obtained herein, the relatively high-poweredlight (e.g., dosed at hundreds of J/cm²) used in the past to disinfectsurfaces cannot safely be used in vivo. Importantly, the dosage of light(J/cm²) had to be sufficiently non-cytotoxic (i.e., would not reduceviability by more than 25% at a dose that resulted in an EC₅₀). Theresulting LTI is expected to vary depending on the type of cell exposedto the phototherapy, but for a given cell type, ideally there is aneffective therapeutic window, such as an LTI of at least 2, or in arange from 2 to 100,000, or in a range from 2 to 1000, or in a rangefrom 2 to 250, depending on the application. Because SARS-CoV-2,influenza and other viruses have lipid membranes, and part of the methodby which the light kills the viruses is believed to be oxidative damageto these membranes, it is believed that this treatment will also workequally well on other respiratory viruses. Further, the treatmentsdescribed herein may also work on viruses that do not have lipidmembranes (e.g., rhinoviruses that cause most common colds).

While the above-described examples are provided in the context of viralapplications, the principles of the present disclosure may also beapplicable for treatment of bacterial infections. There is a currentproblem when treating bacterial respiratory infections, namely, AMR andrecalcitrant lung infections. Antimicrobial resistance has led to manypatients having their lungs infected with bacteria that are resistant tomany common antibiotics. As new antibiotics become developed, bacterialresistance soon follows. One potential solution to this problem would beto use visible light as described herein, at an effective antimicrobialwavelength and dosage, alone or in combination with conventionalantibiotic therapy. While bacteria can develop resistance againstantibiotics, it is more difficult for them to develop resistance toantimicrobial therapy using visible light. The potential uses arefar-reaching; so long as the light is delivered in a safe, therapeuticdosage, patients can be effectively treated for a number of respiratorymicrobial infections, such as tuberculosis, mycobacterium avium complex,and the like, and specifically including those caused by spore-formingbacteria. Bacterial infections caused by spore-forming bacteria can beparticularly difficult to treat with conventional antibiotics, becausethe antibiotics only kill bacteria when they are not in spore form. Asdisclosed herein, certain wavelengths of light are effective at killingspore-forming microbes not only in their active form, but also in theirspore form.

As discussed below, not all light at blue wavelengths are equivalent.Some have higher cytotoxicity to the infected tissues, and some havehigher antimicrobial efficacy. It is useful to consider lighttherapeutic index (LTI), which is a combination of antimicrobialactivity and safety to the exposed tissues. Accordingly, a series ofexperiments were performed to identify suitable wavelengths and dosagelevels to provide safe and effective antibacterial treatments.

For the experiments, bacterial cultures were prepared in 1X phosphatebuffered saline (PBS) or CAMHB at 106 CFU/ml, and 200 μl were aliquotedinto wells of a 96-well microtiter plate. Plates with lids were placedunder a white illumination box, with an LED array placed on top suchthat the light shines down onto bacteria. A fan blew across the devicethough vents in the illumination box to minimize the heat generated bythe LED lights. All setups were done inside a Class II biosafetycabinet. Lights were turned on for a given time, then bacteria weresampled, serially diluted, and plated on MHA for enumeration.

The bacterial strains used in this study were obtained from the AmericanType Culture Collection (ATCC), the CDC-FDA's Antimicrobial ResistanceBank (AR-BANK), from Dr. John LiPuma at the Burkholderia cepaciaResearch Laboratory and Repository (BcRLR) at the University ofMichigan, or from the laboratory of Dr. Mark Schoenfisch at theUniversity of North Carolina Chapel Hill. Strains from the BcRLR wereconfirmed to be Pseudomonas aeruginosa by 16S sequencing, and the otherstrains were confirmed to be P. aeruginosa by growth on Pseudomonasisolation agar. Strains were stored in 20% glycerol stocks at −80° C.Strains were cultured on tryptic soy agar (TSA) at 30° C. or 37° C. for1-2 days, or in cation-adjusted Mueller-Hinton Broth. Streptococcuspyogenes and Haemophilus influenzae were grown using Brain HeartInfusion in a chamber with 5% CO2 packets. All bacteria were incubatedat 37° C. Cytoxicity was measured as described above with respect to theantiviral data.

FIG. 98A is a chart 852 showing the effectiveness of light at 405, 425,450, and 470 nm and administered with a dose of 58.5 J/cm², in terms ofhours post-exposure, at killing P. aeruginosa (CFU/ml). The data showthat, at a wavelength of 405 nm or 425 nm, a 5-log reduction inconcentration was observed almost instantaneously, and the effect wasmaintained for four hours post-exposure.

FIG. 98B is a chart 854 showing the effectiveness of light at 405, 425,450, and 470 nm, and administered with a dose of 58.5 J/cm², in terms ofhours post-exposure, at killing S. aeurus (CFU/ml). The data show that,at a wavelength of 405 nm, a 3-log reduction was observed within a halfhour post-exposure, and this increased to a 4-log reduction by 2 hourspost-exposure. At 425 nm, a 2-log reduction in concentration wasobserved within two hours, and this increased to a 4-log reduction by 4hours post-exposure. At 450 nm, a 2-log reduction in concentration wasobserved within three hours, and this increased to a 4-log reduction by4 hours post-exposure. Light at 470 nm was virtually ineffective.

FIG. 99A is a chart 856 showing the effectiveness of light at 425 nm andadministered with doses ranging from 1 to 1000 J/cm² at killing P.aeruginosa (CFU/ml). The data show that, at a wavelength of 425 nm, atdoses of around 60 J/cm², a 4-log reduction in concentration wasobserved, whereas at doses of 100 J/cm² or higher, a 5-log reduction wasobserved.

FIG. 99B is a chart 858 showing the effectiveness of light at 425 nm andadministered with doses ranging from 1 to 1000 J/cm² at killing S.aureus (CFU/ml). The data show that, at a wavelength of 425 nm, at dosesof around 100 J/cm² or more, a 4-log or even a 5-log reduction inconcentration was observed.

FIG. 100A is a chart 860 showing the effectiveness of light at 405 nmand administered with doses ranging from 1 to 1000 J/cm² at killing P.aeruginosa (CFU/ml). The data show that, at a wavelength of 405 nm, atdoses of around 60 J/cm², a 4-log reduction in concentration wasobserved, whereas at doses of 100 J/cm² or higher, a 5-log reduction wasobserved.

FIG. 100B is a chart 862 showing the effectiveness of light at 405 nmand administered with doses ranging from 1 to 1000 J/cm² at killing S.aureus (CFU/ml). The data show that, at a wavelength of 405 nm, at dosesof around 100 J/cm² or more, a 5-log reduction in concentration wasobserved.

FIG. 101 is a chart 864 showing the toxicity of 405 nm and 425 nm lightin primary human aortic endothelial cells (HAEC). Data is providedshowing the effect of light at 405 nm and at 425 nm for a variety ofindicated doses. Even at dosages up to 99 J/cm², the viability of thecells never dropped below 75%, which is a useful threshold fordetermining the safety of a treatment.

FIG. 102A is a chart 866 showing the bacterial logio reduction and the %loss of viability of infected AIR-100 tissues following exposure of thetissue to doses of light ranging from 4 to 512 J/cm² at 405 nm. FIG.102B is a chart 868 showing the bacterial logio reduction and the % lossof viability of infected AIR-100 tissues following exposure of thetissue to doses of light ranging from 4 to 512 J/cm² at 425 nm. At bothwavelengths (405 nm and 425 nm), notable bacterial logio reductions arerealized before dose levels reach 25% loss in tissue viability.

In a similar manner, additional data as described above for FIGS. 102Aand 102B were collected and provided as shown in FIGS. 102C-102F. Thisdata demonstrates similar results, thereby confirming identification ofsafe and effective operating windows. FIG. 102C is a chart 870 showingthe bacterial logio reduction and the % loss of viability of infectedAIR-100 tissues with gram negative bacteria (e.g., P. aeruginosa)following exposure of the tissue to doses of light ranging from 4 to 512J/cm² at 405 nm. FIG. 102D is a chart 872 showing the bacterial logioreduction and the % loss of viability of infected AIR-100 tissues withgram negative bacteria (e.g., P. aeruginosa) following exposure of thetissue to doses of light ranging from 4 to 512 J/cm² at 425 nm. FIG.102E is a chart 874 showing the bacterial logio reduction and the % lossof viability of infected AIR-100 tissues with gram positive bacteria(e.g., S. aureus) following exposure of the tissue to doses of lightranging from 4 to 512 J/cm² at 405 nm, in a similar manner to FIGS. 102Aand 102C. FIG. 102F is a chart 876 showing the bacterial logio reductionand the % loss of viability of infected AIR-100 tissues with grampositive bacteria (e.g., S. aureus) following exposure of the tissue todoses of light ranging from 4 to 512 J/cm² at 425 nm, in a similarmanner to FIGS. 102B and 102D.

Most in-vitro assays against bacteria are conducted in a cell-freesystem. There are two classic or industry standard measurements foranti-bacterial activity. The first is related to inhibition of growthand may be quantified in terms of a minimum inhibitory concentration(MIC). The MIC refers to the dose required to completely inhibit growthof bacteria over a 24-hour period in a broth/growth medium. Given therapidly dividing nature of bacteria, any growth leads to highconcentration of microorganism. Stated differently a 50% reduction isnot sufficient for bacterial infections. A second standard is related tobactericidal results and may be quantified in terms of a minimumbactericidal concentration (MBC). The MBC refers to the dose required toresult in a 3-log reduction (e.g., 99.9%) of bacteria. Assays can be runin PBS or broth/growth media and lead to different results and time isalso a variable. In general, for the bacterial experiments describedabove, the MIC dose for a given organism has typically been greater thanthe MBC determined in phosphate buffered saline.

FIGS. 103A-103J are a series of charts showing the effect of light at405 nm and 425 nm, at differing dosage levels, in terms of bacterialsurvival (CFU/ml) vs. dose (J/cm²). The data is provided for both P.aeruginosa and S. aureus bacteria. As illustrated, light at 405 nm isparticularly effective at killing these bacteria, and that light at 425nm is also effective, though either not as effective, or not effectiveat higher doses. MBC values are indicated on the charts of FIGS.103A-103J to show 3-log reductions in bacteria.

For the purposes of the present bacterial experiments, LTI calculationsmay be realized from the above-referenced data for providing safe andeffective phototherapeutic treatments. As previously described, LTI maybe determined from the relationship of IC₂₅ divided by the EC₅₀ in thecontext of viruses. For the bacterial data presented in FIGS. 102A-103J,the EC₅₀ values may be replaced or substituted with MBC values asillustrated in FIGS. 103A-103J. The IC₂₅ values may be determined by thehorizontal dashed lines indicating 25% loss of tissue viability in FIGS.102A-102D.

FIG. 104 is a table 878 summarizing the LTI calculations andcorresponding bactericidal doses for the bacterial experimentsillustrated in FIGS. 102A-103J. Notably, the bacterial pathogens areselected as those that are commonly associated with bacterial pneumonia.As illustrated, safe and effective phototherapy treatments for gramnegative P. aeruginosa strains according to this experiment may have LTIvalues in a range from 1.5 to 2.5, thereby indicating LTI values forsuch strains may be provided with values of at least 1.5 or higher. Forgram positive S. aureus strains, the LTI values for this experiment arelower for some of the doses than the P. aeruginosa strains.

FIG. 105 is a chart 880 showing the effect of 425 nm light at variousdoses at killing P. aeuriginosa (CFU/ml) over a period of time from 0hours, 2 hours, 4 hours, and 22.5 hours. At higher doses of light, suchas 120 J/cm², the bacterial concentration actually decreases over time.Importantly, it is largely irrelevant whether the entire dosage of light(J/cm²) is administered in one dose, or in a combination of smallerdoses, so long as the same amount of light is administered before thebacteria rebound.

FIG. 106 is a chart 882 showing that whether all of the light (J/cm²) isadministered in one dose or in a series of smaller doses, theantimicrobial effect (average CFU/ml) vs. dose (J/cm2×number oftreatments) is largely the same, at 8 hours and 48 hourspost-administration.

FIG. 107A is a chart 884 showing the treatment of a variety ofdrug-resistant bacteria (Average CFU/ml) vs. dose (J/cm²) at 24 hourspost-exposure. At doses of 80-120 J/cm² (a combination of two treatmentsof 40, 50, or 60 J/cm²), all of the different drug-resistant bacterialstrains were effectively killed. In this regard, the treatmentsdescribed herein offer advantages over antibiotic treatments, in that a)drug resistance is not observed following treatment, and b) thetreatment can be effective against drug-resistant bacteria. As shown inFIG. 107A, when the treatment was applied to a variety of drug-resistantbacteria, at doses of 80-120 J/cm² in a combination of two treatments of40, 50, or 60 J/cm², all of the different drug-resistant bacterialstrains were effectively killed.

FIG. 107B is a table 886 summarizing the bacteria species and strainsthat were tested. ATCC refers to American Type Culture Collection. BcRLRrefers to Burkholderia cepacia Research Laboratory and Repositoryprovided by Dr. John LiPuma of the University of Michigan. MDR refers tomultidrug resistant, e.g., resistant to 3 classes of antibiotics. XDRrefers to extremely drug resistant, e.g., resistant to 5 classes ofantibiotics, such as amikacin (AMK), aztreonam (ATM), cefepime (FEP),ceftazidime (CAZ), ceftazidime-avibactam (CZA), ceftolozane-tazobactam(C/T), ciprofloxacin (CIP), colistin (CST), doripenem (DOR), gentamicin(GEN), imipenem (IPM), levofloxacin (LVX), meropenem (MEM),piperacillin-tazobactam (TZP), or tobramycin (TOB).

FIG. 107C is a table 888 that summarizes the efficacy of twice dailydosing of 425 nm light against difficult-to-treat clinical lungpathogens. Bactericidal doses are in PBS and for a 3-log reductionrelative to dark control samples. MIC doses are in broth with no changein CFU/ml relative to starting CFU/ml. MBC doses are in broth and for a3-log reduction in CFU/ml relative to dark control samples. Accordingly,one can use the treatments described herein to deliver safe andeffective antimicrobial treatments to a number of different bacterialinfections, including those caused by drug-resistant bacteria.Additionally, illumination devices and treatments as disclosed hereinmay provide multiple pathogenic benefits (e.g., for viruses, bacteria,and fungi) with single wavelength and/or multiple wavelength lighttreatments.

Light therapies as disclosed herein may be combined with conventionalpharmaceutical agents, such as antivirals, anticoagulants,anti-inflammatories, and the like, and the antiviral wavelengths can becombined with anti-inflammatory wavelengths to reduce the inflammatorydamage caused by the virus, by the cytokine storm induced by the virus,and/or by the phototherapy at the antiviralNO-producing/NO-releasing/singlet oxygen producing wavelengths.

It is contemplated that any of the foregoing aspects, and/or variousseparate aspects and features as described herein, may be combined foradditional advantage. Any of the various embodiments as disclosed hereinmay be combined with one or more other disclosed embodiments unlessindicated to the contrary herein.

Those skilled in the art will recognize improvements and modificationsto the preferred embodiments of the present disclosure. All suchimprovements and modifications are considered within the scope of theconcepts disclosed herein and the claims that follow.

What is claimed is:
 1. A method comprising: providing light comprising afirst peak wavelength and a second peak wavelength; and irradiatingmammalian tissue with the light; wherein the first peak wavelengthdiffers from the second wavelength by at least 5 nanometers (nm), thefirst peak wavelength is configured to induce a first biological effect,and the second peak wavelength is configured to induce a secondbiological effect that is different than the first biological effect. 2.The method of claim 1, wherein the first biological effect and thesecond biological effect comprise different ones of inactivating one ormore pathogens that are in a cell-free environment, inhibitingreplication of one or more pathogens that are in a cell-associatedenvironment, upregulating a local immune response, stimulating enzymaticgeneration of nitric oxide to increase endogenous stores of nitricoxide, releasing nitric oxide from endogenous stores of nitric oxide,and inducing an anti-inflammatory effect.
 3. The method of claim 2,wherein the first biological effect comprises inactivating the one ormore pathogens that are in a cell-free environment and inhibitingreplication of the one or more pathogens that are in a cell-associatedenvironment.
 4. The method of claim 3, wherein the first biologicaleffect further comprises upregulating a local immune response.
 5. Themethod of claim 1, wherein the first peak wavelength is in a range from400 nm to 900 nm and the second peak wavelength is in a range from 400nm to 900 nm.
 6. The method of claim 1, wherein the first peakwavelength is in a range from 400 nm to 490 nm and the second peakwavelength is in a range from 490 nm to 900 nm.
 7. The method of claim1, wherein the first peak wavelength is in a range from 400 nm to 490 nmand the second peak wavelength is in a range from 320 nm to 400 nm. 8.The method of claim 1, wherein the first peak wavelength is a range offrom 410 nm to 440 nm.
 9. The method of claim 1, wherein the lightfurther comprises a third peak wavelength that is configured to induce athird biological effect that is different than the first biologicaleffect and the second biological effect, wherein: the first peakwavelength is in a range from 400 nm to 490 nm; the second peakwavelength is in a range from 490 nm to 900 nm; and the third peakwavelength is in a range from 200 nm to 400 nm.
 10. The method of claim1, wherein impinging light to the mammalian tissue comprisesadministering the first peak wavelength in a first time window and thesecond peak wavelength in a second time window.
 11. The method of claim10, wherein the first time window is the same as the second time window.12. The method of claim 10, wherein the first time window is differentthan the second time window.
 13. The method of claim 12, wherein thefirst time window overlaps with the second time window.
 14. The methodof claim 12, wherein the first time window is non-overlapping with thesecond time window.
 15. An illumination device comprising: at least onelight source arranged to impinge light on mammalian tissue within abody, the light comprising at least a first peak wavelength and a secondpeak wavelength and the first peak wavelength differs from the secondwavelength by at least 5 nanometers (nm), wherein the first peakwavelength is configured to induce a first biological effect, and thesecond peak wavelength is configured to induce a second biologicaleffect that is different than the first biological effect; and drivercircuitry configured to drive the at least one light source;
 16. Theillumination device of claim 15, wherein the first biological effect andthe second biological effect comprise different ones of inactivating oneor more pathogens that are in a cell-free environment, inhibitingreplication of one or more pathogens that are in a cell-associatedenvironment, upregulating a local immune response, stimulating enzymaticgeneration of nitric oxide to increase endogenous stores of nitricoxide, releasing nitric oxide from endogenous stores of nitric oxide,and inducing an anti-inflammatory effect.
 17. The illumination device ofclaim 15, wherein the first biological effect comprises inactivating theone or more pathogens that are in a cell-free environment in the bodyand inhibiting replication of the one or more pathogens that are in acell-associated environment in the body.
 18. The illumination device ofclaim 17, wherein the first biological effect further comprisesupregulating a local immune response within the body.
 19. Theillumination device of claim 15, wherein the first peak wavelength is ina range from 400 nm to 490 nm and the second peak wavelength is in arange from 490 nm to 900 nm.
 20. The illumination device of claim 15,wherein the first peak wavelength is in a range from 400 nm to 490 nmand the second peak wavelength is in a range from 320 nm to 400 nm. 21.The illumination device of claim 15, wherein the first peak wavelengthis a range from 410 nm to 440 nm.
 22. The illumination device of claim15, wherein the light further comprises a third peak wavelength that isconfigured to induce a third biological effect that is different thanthe first biological effect and the second biological effect, wherein:the first peak wavelength is in a range from 400 nm to 490 nm; thesecond peak wavelength is in a range from 490 nm to 900 nm; and thethird peak wavelength is in a range from 200 nm to 400 nm.
 23. Theillumination device of claim 15, wherein the at least one light sourcecomprises at least one first emitter that is configured to provide thefirst peak wavelength and at least one second emitter that is configuredto provide the second peak wavelength.
 24. The illumination device ofclaim 23, wherein each of the at least one first emitter and the atleast one second emitter comprises at least one light emitting diode.25. The illumination device of claim 23, wherein the at least one firstemitter comprises a monochromatic light emitting diode and the at leastone second emitter comprises a phosphor-converted light emitting diode.